CN101970000A - Prevention and treatment of cerebral amyloid angiopathy - Google Patents

Abstract

The invention provides improved agents and methods for treatment of cerebral amyloid angiopathy (CAA) and methods to effect prophylaxis of CAA. The methods can treat CAA concurrently with Alzheimer's disease or separately. The methods can effect prophylaxis of CAA concurrently with Alzheimer's disease or separately. The methods involve administering antibody that is specific for the N-terminus of Ass or an agent that can induce such an antibody.

Description

The prevention of cerebral amyloid angiopathy and treatment

The cross reference of related application

Do not have.

Background of invention

The overexpression of mutant human amyloid precursor protein (APP) in various transgenic mices caused the infringement of several Alzheimer's disease (AD) type, and [summary is seen D.Games etc., JAlzheimers Dis 9,133-49 (2006); J.Gotz etc., Mol Psychiatry 9,664-83 (2004)].These comprise essence amyloid beta (A β) speckle, neuritis's pathology, synapse is lost and the development of gliosis.Many reports show that in preclinical study, initiatively (see D.Schenk etc., Nature 400,173-7 (1999); D.L.Dickstein etc., Faseb J20,426-33 (2006)) and passive (see F.Bard etc., Nat Med 6,916-9 (2000); M.Buttini etc., J Neurosci 25,9096-101 (2005); D.M.Wilcock etc., JNeuroinflammation 1,24 (2004)) A β immunotherapy method effectively (sees R.P.Brendza ﹠amp to reducing or eliminating these pathology; D.M.Holzman, Alzheimer Dis Assoc Disord20,118-23 (2006); CA.Lemere etc., Rejuvenation Res 9,77-84 (2006)).In addition, many researchs in various cognitive tests, demonstrate improvement (see D.M.Wilcock etc., the same; C.Janus etc., Nature 408,979-82 (2000); D.Morgan etc., Nature 408,982-5 (2000)).These discoveries have obtained the support (AN1792) of the relevant discovery that obtains from the patient's of selected A β immunization therapy clinical trial recall tests and brain neuroblastoma pathological examination, see J.A.Nicoll etc., Nat Med 9,448-52 (2003); I.Ferrer etc., Brain Pathol 14,11-20 (2004); S.Gilman etc., Neurology 64,1553-62 (2005).

Recently, pathological another the common aspect of AD, blood vessel A β (Va β) has become the theme that studies in great detail of APP transgenic animal preclinical study.Especially, report, passive immunity relevant with VA β with little hemorrhage increase (see D.M.Wilcock etc., the same; M.M.Racke etc., J Neurosci 25,629-36 (2005)).Yet, the clinical meaning that is indicated is still unclear, particularly in view of these identical researchs certain some in favourable consequence (seeing D.M.Wilcock etc., the same), be untreated and handle in the transgenic mice, the vascular morphology of hemosiderin positive vessels is learned the Ultrastructural difference of shortage and (is seen G.J.Burbach etc., Neurobiol Aging 28,202-12 (2007)), and particularly, in ongoing clinical trial, lack the relevant causal evidence of massive hemorrhage or apoplexy.In addition, known little about it by A β immunotherapy method effect to VA β; For example, whether the consequence of chronic treatment pattern tolerance is different with more acute research.For example, although long treatment in fact can prevent or reverse the blood vessel amyloid, whether the VA β increase that still unknowable report is crossed has represented and A β removes relevant transient phenomenon.At last, VA β effect in the transgenic mice also may be according to the APP mutant that is adopted and difference because the relative extent that A β 40 and A β 42 produce influence probably A β aggregation properties and some antibody, especially those have the joint efficiency of the antibody of C-terminal epi-position.

Summary of the invention

The invention provides the method for therapeutic treatment CAA.This method comprises to suffering from or suspecting that the patient who suffers from CAA uses the medicament of effective scheme.In certain methods, thereby this medicament is the antibody that the N-terminal of A β is had specificity and treatment patient.Optional, this medicament is the intra-residue antibody in 1-5 position that is combined in A β.Optional, this antibody is humanization, people or chimeric antibody.Optional, this humanized antibody is 3D6.Optional, this 3D6 humanized antibody is a Ba Biniu pearl monoclonal antibody (bapineuzumab).Optional, this humanized antibody is 12A11.

In certain methods, this medicament is the fragment of A β.Optional, this fragment starts from 1 residue of A β, finally one of 5-10 position residue of A β.Optional, this fragment is A β 1-7.Optional, this A β fragment is with pharmaceutically useful adjuvant administration.Optional, this A β fragment induces the carrier at this segmental antibody to link to each other with helping this fragment.Optional, this carrier links to each other with the segmental C-terminal of A β.

Certain methods of the present invention further comprises determines that the patient suffers from CAA, and wherein determining step took place before dosing step.In certain methods, determining step determines that the patient suffers from the clinical symptoms of CAA.

In the certain methods of therapeutic treatment CAA, lack the characteristic speckle of Alzheimer's disease in patient's brain.Optional, lack the characteristic speckle of Alzheimer's disease and the symptom that the patient lacks Alzheimer's disease in patient's brain.In the certain methods of therapeutic treatment CAA, existing heart attack of patient or apoplexy.

Optional, this method comprises that application dosage is about antibody of 0.01 to about 5mg/kg.Optional, this method comprises that application dosage is about antibody of 0.1 to about 5mg/kg.Optional, this method comprises the dosage of using about 0.5mg/kg.Optional, this method comprises the dosage of using about 1.5mg/kg.Optional, this method comprises uses about dosage of 0.5 to about 3mg/kg.Optional, this method comprises uses about dosage of 0.5 to about 1.5mg/kg.Optional, this method comprises repeatedly administration of antibodies.Optional, this antibody is weekly to per season single administration.Optional, per 13 weeks of this antibody are administered once.Optional, this antibody is through intravenous or subcutaneous administration.

Optional, thus antibody is according to being enough to make the average serum concentration of patient's antibody maintain the scheme administration of also treating the patient in the scope of 1-15 μ g antibody/ml serum.Optional, average serum concentration is in the scope of 1-10 μ g antibody/ml serum.Optional, average serum concentration is in the scope of 1-5 μ g antibody/ml serum.Optional, average serum concentration is in the scope of 2-4 μ g antibody/ml serum.Optional, antibody is according to being enough to make the average serum concentration of antibody keep the scheme administration at least one year.Optional, the average serum concentration of antibody was kept 6 months at least.

At medicament is in the certain methods of antibody, the optional concentration of measuring antibody in the serum that further comprises, and if the concentration that records drop on outside the scope, then carry out project setting.At medicament is in the certain methods of antibody, the optional concentration of measuring antibody in the serum that further comprises, and if the concentration that records drop on outside the scope, then carry out project setting.

Optional, thus antibody is according to being enough to make the average serum concentration of patient's antibody maintain interior patient's the scheme of also treating of scope of 1-15 μ g antibody/ml serum through intravenous administration.Optional, one time every month intravenous is used the dosage of 0.5-1.0mg/kg.Optional, one time every month intravenous is used the dosage of 0.1-1.0mg/kg.

Optional, antibody is through subcutaneous administration.Optional, antibody with weekly to mensal frequency through subcutaneous administration.Optional, antibody is subcutaneous administration biweekly once in a week or whenever.Optional, antibody is with about 0.01 to about 0.35mg/kg dosage subcutaneous administration.Optional, antibody is with about 0.05 to about 0.25mg/kg dosage subcutaneous administration.Optional, antibody is with about dosage of 0.015 to about 0.2mg/kg extremely every subcutaneous administration biweekly once in a week.Optional, antibody is with about dosage of 0.05 to about 0.15mg/kg extremely every subcutaneous administration biweekly once in a week.Optional, antibody is with the weekly subcutaneous administration of about dosage of 0.05 to about 0.07mg/kg.Optional, the antibody warp is with the weekly subcutaneous administration of the dosage of 0.06mg/kg.Optional, antibody is with the every subcutaneous administration biweekly of about dosage of 0.1 to about 0.15mg/kg.

Optional, antibody is with about dosage of 0.01 to about 0.6mg/kg and weekly to mensal frequency subcutaneous administration.Optional, antibody is with about 0.05 to about 0.25mg/kg dosage subcutaneous administration.Optional, antibody is with about dosage of 0.015 to about 0.2mg/kg extremely every subcutaneous administration biweekly once in a week.Optional, antibody is with about dosage of 0.05 to about 0.15mg/kg extremely every subcutaneous administration biweekly once in a week.Optional, antibody is with the weekly subcutaneous administration of about dosage of 0.05 to about 0.07mg/kg.Optional, antibody is with the weekly subcutaneous administration of the dosage of 0.06mg/kg.Optional, antibody is with about 0.1 to about 0.15mg/kg dosage per two all subcutaneous administrations.Optional, antibody is with one time every month subcutaneous administration of about dosage of 0.1 to about 0.3mg/kg.Optional, antibody was administered once with the dosage of 0.2mg/kg in every month.

In certain methods of the present invention, further comprise the variation of the S or S of monitoring CAA response dosing step.Certain methods of the present invention further comprises second medicament of using effective treatment CAA.

The invention provides the method that realizes the CAA prevention.This method comprises that the patient to easy trouble CAA uses the medicament of effective scheme.This medicament is that the N-terminal to A β has specific antibody, and perhaps, this medicament is being induced such antibody after patient's administration, thereby realizes patient's prevention.The invention provides medicament application in the Alzheimer's disease in treatment or prevention, wherein said medicament is that the N-terminal to A β has specific antibody or inducing such antibody after patient's administration.

The invention provides the method for the blood vessel amyloid that reduces the patient.This method comprises that the N-terminal of using A β according to effective removal blood vessel amyloid and the relevant therapeutic scheme of the minimizing little hemorrhage incidence rate of brain has specific antibody.Certain methods further comprises by MRI monitoring patient's brain little hemorrhage.Certain methods further comprises the scanning by PET, the removal of monitoring patient's blood vessel amyloid.Optional, in certain methods, therapeutic scheme is a long-term treatment regimen.Optional, in certain methods, therapeutic scheme comprises 0.01-5mg/kg weight in patients and weekly antibody dosage to per season single administration.Optional, in certain methods, the dosage of antibody is 0.1-5mg/kg.Optional, in certain methods, dosage is about 0.5mg/kg.Optional, in certain methods, dosage is about 1.5mg/kg.Optional, in certain methods, dosage is about 0.5 to about 3mg/kg.Optional, in certain methods, dosage is about 0.5 to about 1.5mg/kg.Optional, in certain methods, per 13 weeks of dosage use once.Optional, in certain methods, antibody is through intravenous or subcutaneous administration.Optional, medicament is the intra-residue antibody in 1-5 position that is combined in A β.Optional, antibody is humanization, people or chimeric antibody.Optional, this humanized antibody is 3D6.Optional, the 3D6 humanized antibody is a Ba Biniu pearl monoclonal antibody (bapineuzumab).Optional, humanized antibody is 12A11.

The invention provides the method for treatment Alzheimer's disease.This method comprises: to reduce or to suppress development that the blood vessel amyloidogenic becomes (amyloidogenic) pathology, to make little hemorrhage minimizedly and/or reduce or suppress the pharmaceutical quantities of the development of A beta plaque, the N-terminal of using A β has specific antibody.Optional, in certain methods, antibodies is in the residue of the 1-5 position of A β.Optional, antibody is humanization, people or chimeric antibody.Optional, humanized antibody is 3D6.Optional, the 3D6 humanized antibody is a Ba Biniu pearl monoclonal antibody (bapineuzumab).Optional, humanized antibody is 12A11.

The invention provides the method for treatment Alzheimer's disease, it comprises: to reduce or to suppress development that the blood vessel amyloidogenic becomes pathology, to make little hemorrhage minimizedly and/or reduce or suppress the dosage of the development of neuritis's pathology, the N-terminal of using A β has specific antibody.Optional, in certain methods, antibodies is in the residue of the 1-5 position of A β.Optional, antibody is humanization, people or chimeric antibody.Optional, humanized antibody is 3D6.Optional, the 3D6 humanized antibody is a Ba Biniu pearl monoclonal antibody (bapineuzumab).Optional, humanized antibody is 12A11.

The invention provides the method for treatment Alzheimer's disease, it comprises: become pathology, make little hemorrhage minimizedly and/or improve the dosage of patient's cognitive function to reduce or to suppress the blood vessel amyloidogenic, the N-terminal of using A β has specific antibody.Optional, in certain methods, antibodies is in the residue of the 1-5 position of A β.Optional, antibody is humanization, people or chimeric antibody.Optional, humanized antibody is 3D6.Optional, the 3D6 humanized antibody is a Ba Biniu pearl monoclonal antibody (bapineuzumab).Optional, humanized antibody is 12A11.

Optional, in the method for some treatment Alzheimer's disease, minimizing or inhibition blood vessel amyloidogenic change pathology are to the cumulative prevention of blood vessel A β or remove blood vessel A β.

The present invention further provides the diagnostic kit that is applicable to said method.Such test kit comprises and the bonded antibody of epitope specificity with 1-10 position residue of A β.Some test kits have label, and the usage in the Alzheimer's disease is diagnosed or monitored to description antibody in vivo.

The present invention further provides the kit of the treatment CAA that is applicable to said method.Such kit comprises the vial that contains preparation.Kits more of the present invention comprise the vial that contains preparation, and said preparation comprises about 0.5 to 3mg/kg humanization anti-amyloid beta antibodies.Kits more of the present invention comprise the vial that contains preparation, and said preparation comprises: the about 10mg of i. is about 250mg humanization anti-amyloid beta antibodies extremely, about 4% mannitol of ii. or about 150mM NaCl, and the about 5mM of iii. is to about 10mM histidine and the about 10mM methionine of iv..Some kits comprise the description of the patient's who monitors administered formulation CAA.Optional, description comprises: i. is little hemorrhage by MRI monitoring patient's brain, or ii. is by the removal of PET scanning monitoring patient's blood vessel amyloid.

The present invention further provides the kit of the treatment Alzheimer's disease that is applicable to said method.Such kit comprises the vial that contains preparation, and said preparation comprises: the about 10mg of i. is about 250mg humanization anti-amyloid beta antibodies extremely, about 4% mannitol of ii. or about 150mM NaCl, and the about 5mM of iii. is to about 10mM histidine and the about 10mM methionine of iv..Some kits contain the description of the patient's who monitors administered formulation Alzheimer's disease.Optional, description comprises: i. is little hemorrhage by MRI monitoring patient's brain, or ii. is by the removal of PET scanning monitoring patient's blood vessel amyloid.

The treatment CAA that is applicable to said method and the kit of Alzheimer's disease have been the present invention further provides.Such kit comprises the vial that contains preparation, and said preparation comprises: the about 10mg of i. is about 250mg humanization anti-amyloid beta antibodies extremely, about 4% mannitol of ii. or about 150mMNaCl, and the about 5mM of iii. is to about 10mM histidine and the about 10mM methionine of iv..Some kits comprise that monitoring system uses patient's the CAA of medicament and the description of Alzheimer's disease.Optional, description comprises: i. is little hemorrhage by MRI monitoring patient's brain, or ii. is by the removal of PET scanning monitoring patient's blood vessel amyloid.

Optional, antibody is with about dosed administration of 0.05 to about 0.5mg/kg.Optional, antibody is with about dosage of 1 to about 40mg and weekly to mensal frequency administration.Optional, antibody is with about dosage of 5 to about 25mg and weekly to mensal frequency administration.Optional, antibody is with about dosage of 2.5 to about 15mg and weekly to mensal frequency administration.

Optional, antibody is weekly to administration biweekly whenever with about dosage of 1 to about 12mg.Optional, antibody is weekly to administration biweekly whenever with about dosage of 2.5 to about 10mg.Optional, antibody is administered once weekly with about dosage of 2.5 to about 5mg.Optional, antibody is administered once weekly with about dosage of 4 to about 5mg.Optional, antibody was administered once with about two weeks of 7 to about 10mg dosage.

The accompanying drawing summary

Fig. 1 a has shown that thioflavin S dyeing, Fig. 1 b have shown that the 3D6 of A β in the brain center line blood vessels of 18 monthly age PDAPP mices is immune labeled.What Fig. 1 c showed is people AD tissue, and Fig. 1 d shows is the blood vessel with pia arachnoid and shallow table essence in the PDAPP mice of the immune labeled VA β of 3D6.Scale=100 μ m.

Fig. 2 a shown untreated 12 the monthly age mice brain, Fig. 2 b has shown the mouse brain through control treatment, Fig. 2 c has shown the mouse brain that the immune labeled VA β of 3D6 is arranged in the brain of the mice that 3mg/kg 3D6 handles and Fig. 2 d have shown, center line blood vessel 266 that handle through 3mg/kg.Scale=50 μ m.Figure has shown that each has the percent of nothing to the animal of a little VA β (white rod) and moderate VA β (reticule rod) in organizing.

Fig. 3 a has shown the brain through control treatment, and Fig. 3 b has shown the brain of handling through 0.1mg/kg 3D6, and Fig. 3 c has shown through the brain that 0.3mg/kg 3D6 handles and Fig. 3 d and shown the brain of handling through 3mg/kg3D6, the immune labeled VA β of 3D6 is arranged in the pia arachnoid blood vessel.Parantheses and arrow are represented VA β, scale=100 μ m.Figure has shown that each has the percent of nothing to the animal of a little VA β (white rod) and moderate VA β (crossbar) in organizing.

Fig. 4 a has shown that the circular block of the complete VA β of the pia arachnoid blood vessel that encirclement is suffered from and the 3D6 of band are immune labeled in the mice of 0.1mg/kg 3D6 processing.Fig. 4 b has shown that the 3D6 of the VA β that the fritter in the part reset procedure is corroded is immune labeled in the mice of 0.1mg/kg3D6 processing.Scale=50 μ m.

Fig. 5 a and 5b show, when the dosage of 3D6 was low, the VA β of most of animals was partly removed or stops and do not have little hemorrhage sign.When Fig. 5 c was presented at 3mg/kg 3D6, the VA β of most of animals was removed fully or stops and do not have little hemorrhage sign.Fig. 5 d and 5e show that part was removed the little hemorrhage of position when 3D6 dosage was low.When Fig. 5 f is presented at 3mg/kg 3D6, remove the little hemorrhage of position fully.The arrow points macrophage.Scale=100 μ m.

Fig. 6 a has shown that the hemosiderin of contrast and processed group is evaluated in the A research.Fig. 6 b has shown the contrast in the B research and the hemosiderin evaluation of processed group.

Definition

Two peptide sequences of term " basic homogeneity " expression are by for example GAP or BESTFIT program, when adopting default gap weighting (default gap weights) to carry out the optimum contrast, total at least 65% sequence homogeneity, preferred at least 80% or 90% sequence homogeneity, more preferably at least 95% sequence homogeneity or higher (for example, 99% sequence homogeneity or higher). Preferred not identical residue position is to replace different because of conserved amino acid.

For sequence relatively, usually with a sequence as reference sequence, cycle tests is compared with it. When adopting sequence comparison algorithm, will test and reference sequence input computer, specify the subsequence coordinate, in case of necessity specified sequence algorithm routine parameter. Sequence comparison algorithm calculates cycle tests with respect to the percentage sequence homogeneity of reference sequence according to the program parameter of appointment then.

The optimum contrast of sequence comparison for example can be adopted, Smith and Waterman, the local clustalw algorithm of Adv.Appl.Math.2:482 (1981), Needleman and Wunsch, the homology contrast algorithm of J.Mol.Biol.48:443 (1970), Pearson and Lipman, the similarity searching method of Proc.Nat ' l.Acad.Sci.USA 85:2444 (1988), (the GAP in the Wisconsin science of heredity software kit is implemented in computerization by these algorithms, BESTFIT, FASTA and TFASTA, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (generally seeing Ausubel etc., the same) undertaken. An example that is applicable to the algorithm of determining percentage sequence homogeneity and sequence similarity is the BLAST algorithm, and it is at Altschul etc., and J.Mol.Biol.215:403-410 has description in (1990). The software that carries out the BLAST analysis can openly obtain by national biotechnology center (NCBI) website. Although also can use custom parameter, carry out usually using when sequence compares the default procedures parameter. For amino acid sequence, the BLASTP program employed default be that word length (W) is 3, expectation (E) value be 10 and BLOSUM62 score matrix (referring to, Henikoff and Henikoff, Proc.Natl.Acad.Sci.USA89:10915 (1989)).

For 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor being categorized as conservative or nonconservative purpose, amino acid is grouped as follows: I organizes (hydrophobic side chain): nor-leucine, met, ala, val, leu, ile; II organizes (neutral hydrophilic side chain): cys, ser, thr; III organizes (acid side-chain): asp, glu; IV organizes (basic side chain): asn, gln, his, lys, arg; V organizes (affecting the residue of chain orientation): gly, pro; Organize (aromatic series side chain) with VI: trp, tyr, phe. Conservative replacement comprises with the replacement between amino acid. Non-conservative replacement is exchanged to consist of by the member of one of these classifications and another kind of member.

It is basically pure that healing potion of the present invention is generally, no noxious pollutant. This expression medicament has the purity at least about 50%w/w (w/w) usually, and does not substantially disturb albumen and pollutant. Sometimes, medicament has at least about 80%w/w, more preferably at least about 90% or the purity of about 95%w/w. Yet, adopt conventional protein purification technology, can be obtained up to the homogeneity peptide of few about 99%w/w.

The phrase of molecule and target " specific binding " or " generation specific immune response " refers in the presence of the heterogeneous population of other biological agent, determines the association reaction that this molecule exists. Therefore, under the immunoassay condition of appointment, special molecule preferentially is combined with particular target, and other biological agent that does not exist in sample is combined with significant quantity. Antibody need to be selected antibody for the specificity of target with the specific binding of target under such condition. There are many kinds of immunoassay forms can be used for selecting antibody with specific protein generation specific immune response. For example, conventionally select monoclonal antibody with albumen generation specific immune response with solid phase ELISA immunoassay. About being used for measuring the immunoassay form of specific immune response and the description of condition, referring to, for example, Harlow and Lane (1988) Antibody, A Laboratory Manual (antibody: laboratory manual), Cold Spring Habor Publications, New York. Specific binding between the entity means that affinity is at least 10⁶、10 ⁷、10 ⁸、10 ⁹M ^-1Or 10¹⁰ M ^-1 Greater than 10⁸M ^-1Affinity be preferred.

Term " antibody " or " immunoglobulin (Ig) " are used for comprising complete antibody and binding fragment thereof. Usually, fragment and their complete antibody competition and the specific binding of antigen fragment of deriving comprise independent heavy chain, light chain Fab, Fab ' F (ab ') 2, Fabc and Fv. Fragment is passed through recombinant DNA technology, or is produced by enzyme or the Chemical Decomposition of complete immunoglobulin (Ig). Term " antibody " also comprises one or more with other albumen chemical bond or is expressed as immunoglobulin chain with the fusion of other albumen. Term " antibody " also comprises bispecific antibody. Bispecific or bifunctional antibody be have two differences heavy/light chain to the artificial hybridization antibody of two different binding sites. Bispecific antibody can produce by the connection of several different methods, the fusion that comprises hybridoma or Fab ' fragment. Referring to, for example, Songsivilai and Lachmann, Clin.Exp.Immunol.79:315-321 (1990); Kostelny etc., J.Immunol.148,1547-1553 (1992).

APP ⁶⁹⁵、APP ⁷⁵¹And APP⁷⁷⁰Refer to respectively the long-chain polypeptide by 695,751 and 770 amino acid residues of people's app gene coding. See Kang etc., Nature 325,773 (1987); Ponte etc., Nature 331,525 (1988); With Kitaguchi etc., Nature 331,530 (1988). Amino acid in the human amyloid precursor protein (APP) is specified numbering according to the sequence of APP770 isotype.

Refer to contain the A β peptide of 1-39,1-40,1-41,1-42 and 1-43 amino acids residue such as the term of A β 39, A β 40, A β 41, A β 42 and A β 43. Hardy etc., TINS 20, Fig. 1 of 155-158 (1997) illustrated these peptides sequence and with the relation of APP precursor. For example, A β 42 has sequence:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAHGLMVGGVVIAT| 

A β 41, A β 40 lack respectively Ala, Ala-Ile and Ala-Ile-Val with A β 39 from the different C of the being ends of A β 42. A β 43 exists the Thr residue from the different C of the being ends of A β 42.

" antigen " is the entity of antibody and its specific binding.

Term " epi-position " or " antigenic determinant " refer to the site on the antigen of B and/or t cell response. The B cell epitope can form by adjacency amino acid or because of three grades of protein folding and arranged side by side non-adjacent amino acid. Usually kept after being exposed to the sex change solvent by the amino acids formed epi-position of adjacency, and usually lost after processing with the sex change solvent by three grades of epi-positions that fold and form. Epi-position generally includes with unique space conformation at least 3, more generally at least 5 or 8-10 amino acid. The method of measuring the space conformation of epi-position comprises, for example, and X-ray crystallography and two dimensional NMR. Referring to, for example, Epitope Mapping Protocols in Methods in MolecularBiology (the epitope mapping rules in the molecular biology method), Vol.66, Glenn E.Morris writes (1996). Identifying the antibody of identical epi-position can identify in the simple immunoassay that shows the another kind of antibody of a kind of antibody blocking and target antigen binding ability. About 9 amino acid of T cell recognition cd8 cell or about 13-15 of cd4 cell amino acid whose continuous epitope. The T cell of identification epi-position can differentiate that its mensuration is to mix by the t cell response epi-position of contacted antigen by the analyzed in vitro of measuring antigen dependence propagation³H-thymidine (Burke etc., J.Inf.Dis.170,1110-19 (1994)), kill and wound (cytotoxic T lymphocyte analysis, Tigges etc., J.Immunol.156,3901-3910) or the secretion by cell factor by the antigen dependence.

It is to set up body fluid (by antibody-mediated) and/or cell (by T cells with antigenic specificity or the mediation of its secretory product) for receptor patient's 4 amyloid to reply that term " immunologic " or " immunity " are replied. Replying like this can be to reply by using the active that immunogene induces, or replied by the passive of induced t cell of administration of antibodies or contacted antigen. Cellullar immunologic response is by the initiation of presenting of the polypeptide epitope relevant with I class or II class MHC molecule, with active antigen specific C D4⁺T helper cell and/or CD8⁺Cytotoxic T cell. Reply the activation of other component that also can comprise monocyte, macrophage, NK cell, basophilic granulocyte, dendritic cells, astrocyte, microglia, eosinophil or inherent immunity. The existence of cell-mediated immune response can be passed through proliferation assay (CD4⁺The T cell) or CTL (cytotoxic T lymphocyte) assay determination (see Burke, the same; Tigges, the same). Cell and humoral response can be by isolating respectively antibody and T cell to the Relative Contribution of immunogenic protection or therapeutic action from the syngeneic animal of immunity, and measure protection or therapeutic action is distinguished in second object.

" immunogenic agents " or " immunogene ", optional and adjuvant associating can be induced the immune response of resisting himself after being applied to mammal.

Term " fully-D " refers to have 〉=75%, 〉=80%, 〉=85%, 〉=90%, 〉=95% or 100% the amino acid whose peptide of D-form.

Term " naked polynucleotides " refers to not compound with colloidal substance polynucleotides. Naked polynucleotides are cloned in plasmid vector sometimes.

Term " adjuvant " refers to antigen combined administration the time to strengthen to the immune response of antigen but when individually dosed and the compound of non-confrontational original living immune response. Adjuvant can pass through several mechanisms, comprises raising lymphocyte, stimulation B and/or T cell and stimulating expression of macrophage, strengthens immune response.

The definition of term " effective dose " is to be enough to reach or at least part of amount that produces a desired effect. Term " treatment effective dose " is defined as to be enough to cure or the ill patient's of at least part of containment disease and the amount of complication thereof. The effective dose of this application will depend on the severity of infection and the general state of patient's autoimmunity system.

Owing to for example measure error, some deviations of disclosed scope are admissible. These deviations are shown by the term " about " about scope or dosage.

Term " patient " comprises people and other mammalian object of accepting preventative or therapeutic treatment.

Competition between antibody is by wherein measuring for the analysis of the specific binding that tries immunoglobulin (Ig) inhibition reference antibody and common antigen such as A β. Known have a very eurypalynous competitive binding analysis, for example: the direct or indirect radiommunoassay of solid phase (RIA), the direct or indirect EIA enzyme immunoassay of solid phase (EIA), sandwich competition analysis (seeing Stahli etc., Methods in Enzymology9:242-253 (1983)); The direct biotin-avidin EIA of solid phase (seeing Kirkland etc., J.Immunol.137:3614-3619 (1986)); The direct labeled analysis of solid phase, the direct mark sandwich assay of solid phase (seeing Harlow and Lane, Antibodies, A Laboratory Manual (antibody, laboratory manual), Cold Spring Harbor Press (1988)); (see Morel etc., Molec.Immunol.25 (1): 7-15 (1988): the direct biotin-avidin EIA of solid phase (Cheung etc., Virology 176:546-552 (1990)) with the direct mark RIA of the solid phase of I-125 mark; And direct mark RIA (Moldenhauer etc., Scand.J.Immunol.32:77-82 (1990)). Usually, such analysis relate to use be loaded with unmarked for examination immunoglobulin (Ig) or mark reference immunoglobulin (Ig) any the surface of solids or the purifying antigen of Cell binding. There is the labelled amount lower and surface of solids or Cell binding by being determined at for the examination immunoglobulin (Ig), measures competitive the inhibition. For the common excessive existence of examination immunoglobulin (Ig). The antibody (competitive antibody) of differentiating by competition analysis comprises the antibody identical with the epi-position of reference antibody combination, and with the antibody of the epi-position of fully being combined near reference antibody with the contiguous epi-position combination that produces steric hindrance. Usually, competitive antibody is excessive when existing, and it will suppress at least 50% or 75% reference antibody and the specific binding of common antigen.

Term " symptom " or " clinical symptoms " refer to the subjectivity foundation of the disease of patient's perception, change such as gait. " sign " refers to the objective basis by the disease of doctor's observation.

The compositions or the method that " comprise " one or more described key elements can comprise the key element that other is not specifically told about.

Detailed Description Of The Invention

I. summation

The invention provides the method that realizes prevention and treatment cerebral amyloid angiopathy (CAA), CAA is to be the disease of feature there to be A β peptide blood vessel deposit.These blood vessel deposits are different with the significant essence deposit of Alzheimer's disease.Most of Alzheimer's disease patients are the slight at least CAA of patient.Yet CAA also can be independent of the symptom of Alzheimer's disease and/or characteristic pathology and take place.CAA also with Alzheimer's disease usually without symptom, as apoplexy.The invention provides the method that realizes prevention or treatment CAA, no matter it is independent or concurrent Alzheimer's disease.In the patient of concurrent Alzheimer's disease and CAA, this method can be treated this two kinds of diseases simultaneously.In not suffering from the patient of any disease, this method can realize the prevention of these two kinds of diseases.Do not have suffering from CAA among the patient of Alzheimer's disease, this method can be treated CAA, and realizes the prevention of Alzheimer's disease.This method comprises initiatively or passive immunotherapy.In passive immunotherapy, use the bonded antibody of the intra-residue epi-position in 1-10 position with A β.In active immunity treatment, use medicament, such as the A β fragment that can induce such antibody.Although understanding mechanism is not essential for enforcement of the present invention, think that described antibody combines with A β blood vessel deposit, thereby has promoted sedimental removing.

II. medicament

The medicament that this method is used or at the antibody (passive administration) of the N-terminal of A β perhaps can induced such antibody after patient's administration.Such medicament is existing describe (seeing WO 98/25386 and WO 00/72880) in the science of the immunization therapy of relevant Alzheimer's disease and patent documentation before.

A. active immunity treatment

A β also claims beta-amyloyd peptide or A4 peptide (to see US 4,666,829; Glenner and Wong, Biochem.Biophys.Res.Commun.120,1131 (1984)), be 39-43 amino acid whose peptide, be the main component of the characteristic speckle of Alzheimer's disease.A β is produced (see Hardy, TINS 20,154 (1997)) by these two kinds of enzyme bigger protein A PP of processing that are known as β and gamma secretase.Known mutations occurs in the site of next-door neighbour β or gamma secretase among the APP relevant with Alzheimer's disease, or in A β.For example, 717 sites of A β is processed in the APP cracking with gamma secretase are pressed close to, the cracking site of 670/671 next-door neighbour's beta-secretase.It is believed that, thereby sudden change causes AD by the amount that the lytic response interaction with formation A β increases the A β of 42/43 amino acid form that is produced.

A β has the peculiar property that can fix and activate classical and alternate complement cascade.Especially, it combines with Clq, combines with C3bi at last.This association has promoted and the combining of macrophage, has caused the activation of B cell.In addition, C3bi decomposes, then with the B cell on CR2 combine in T cell dependency mode, cause the activation of these cells to be 10,000 times of increases.This mechanism causes A β to produce above other antigenic immunne response.

Initiatively the preferred agents of administration is the fragment that starts from 1 residue of A β and one of terminate between the residue of 5-10 position.Such fragment can be induced the bonded antibody of N-terminal specificity with A β when being connected with the carrier that suits.That such fragment lacks is naturally occurring, relevant with adverse side effect in the clinical trial of complete A β self t cell epitope.Preferred immunogenic fragments comprises A β 1-5,1-6 and 1-7,1-10,3-7,1-3, and 1-4.The title of A β 1-5, for example,, the expression fragment comprises the 1-5 position residue of A β and lacks other residue of A β.

A β is deutero-to spread part (ADDLs), ADDL succedaneum, ADDL binding molecule and also can be used for active immunity treatment.Referring to, for example, WO 2004/031400, and it draws in full for all purposes and is reference.

Optional, the fragment of A β combines with carrier, induces at this segmental antibody with help.Some medicaments of induce immune response contain and are useful on the suitable epi-position of inducing the immunne response of amyloid, but too little, do not have immunogenicity.In the case, peptide based immunogens can be connected with the carrier molecule that suits, form the conjugate that helps to cause immunne response.Suitable carrier comprises serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, Elityran, ovalbumin, tetanus toxoid, or from other pathogen such as diphtheria (for example, CRM 197), the toxoid of escherichia coli (E.coli), cholera or helicobacter pylori (H.pylori), or the toxin derivant of attenuation.T cell epitope also is the carrier molecule that suits.Some conjugates can pass through medicament of the present invention and immunostimulation polymer molecule (for example, three palmityls-S-glyceryl Cysteine (Pam ₃Cys), mannan (mannose polymer) or glucosan (β 1 → 2 polymer)), cytokine (for example, IL-1, IL-1 α and β peptide, IL-2, γ-INF, IL-10, GM-CSF) and chemokines (for example, MIP1 α and β, and RANTES) connect and form.Can also be as O ' Mahony, described in WO 97/17613 and the WO 97/17614, the peptide of immunogenic agents and enhancing being striden the tissue transhipment links to each other.Immunogen can (for example, carrier gly-gly) connects with being with or without interval aminoacid.

Some conjugates can form by medicament of the present invention is connected with at least one t cell epitope.Some t cell epitopes are non-kind system optionally (promiscuous), and other t cell epitope then is general (universal).Non-kind is that the selectivity t cell epitope can strengthen the inducing T cell immunity in the varied object that shows various HLA types.With non-kind is that the selectivity t cell epitope is opposite, Universal T-cell epitopes can big percentage ratio, for example at least 75%, show in the object by the various HLA molecules of different HLA-DR allele codings, strengthen the inducing T cell immunity.

Naturally occurring t cell epitope has many, as, tetanus toxoid (for example, P2 and P30 epi-position), hbs antigen, DT-Pa, Measles virus F albumen, chlamydia trachomatis (Chlamydia trachomitis) major outer membrane albumen, diphtheria toxoid, Plasmodium falciparum (Plasmodium falciparum) circumsporozoite protein T, Plasmodium falciparum CS antigen, schistosoma mansoni (Schistosoma mansoni) phosphotriose isomerase, Escherichia coli (Escherichia coli) TraT and influenza virus hemagglutinin (HA).Immunogenic peptide of the present invention can also with Sinigaglia F. etc., Nature, 336:778-780 (1988); Chicz R.M. etc., J.Exp.Med., 178:27-47 (1993); Hammer J. etc., cell 74:197-203 (1993); Falk K. etc., Immunogenetics, 39:230-242 (1994); WO 98/23635; With J.Immunology such as Southwood S., the t cell epitope combination of describing among the 160:3363-3373 (1998), above-mentioned each piece of writing all is incorporated herein by reference for all purposes.Other example comprises:

Influenza hemagglutinin: HA _307-319

Malaria CS:T3 epi-position EKKIAKMEKASSVFNV

Hepatitis B surface antigen: HbsAg _19-28FFLLTRILTI

Heat shock protein 65:hsp65 _153-171DQSIGDLIAEAMDKVGNEG

Bacillus calmette-guerin vaccine: QVHFQPLPPAVVKL

Tetanus toxoid: TT _830-844QYIKANSKFIGITEL

Tetanus toxoid: TT _947-967FNNFTVSFWLRVPKVSASHLE

HIV?gp120?T1：KQIINMWQEVGKAMYA

Some examples of conjugate comprise:

AN90549 (the A β 1-7-tetanus toxoid 830-844 of MAP4 configuration):

DAEFRHD-QYIKANSKFIGITEL

AN90550 (the A β 1-7-tetanus toxoid 947-967 of MAP4 configuration):

DAEFRHD-FNNFTVSFWLRVPKVSASHLE

AN90542 (the A β 1-7-tetanus toxoid 830-844+947-967 of linear configuration):

DAEFRHD-QYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE

PADRE peptide (all being linear configuration), wherein X is preferably Cyclohexylalanine, tyrosine or phenylalanine, most preferably is Cyclohexylalanine:

AN90562(PADRE-Aβ1-7)：

AKXVAAWTLAAA-DAEFRHD?

AN90543(3PADRE-Aβ1-7)：

-AKXVAAWTLKAAA

Other example of fusion rotein (the immunogenicity epi-position of A β marks with runic) comprising:

AKXVAAWTLKAAA-DAEFRHD-DAEFRHD-DAEFRHD?

DAEFRHD-AKXVAAWTLKAAA

DAEFRHD-ISQAVHAAHAEINEAGR

FRHDSGY-ISQAVHAAHAEINEAGR

EFRHDSG-ISQAVHAAHAEINEAGR

PKYVKQNTLKLAT-?

DAEFRHD-PKYVKQNTLKLAT-DAEFRHD?

-PKYVKQNTLKLAT

DAEFRHD-DAEFRHD-PKYVKQNTLKLAT

DAEFRHD-PKYVKQNTLKLAT-EKKIAKMEKASSVFNV-QYIKANSKFIGITEI

FNNFTVSFWLRVPKVSASHLE-?

QYIKANSKFIGITELNNFTVSFWLRVPKVSASHLE

DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE

DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-DAEFRHD

On the two collateralization resins

-QYIKANSKFIGITEL

The fragment of A β as A β 1-6, combines with the subunit of carrier such as virus-like particle (VLPs) and VLPs, induces at this segmental antibody with help.Referring to, for example, WO 2004/016282 and US 20040141984, it is incorporated herein by reference in full for all purposes separately.

B. passive immunotherapy

Passive immunotherapy uses the specific antibody of A β N-terminal to finish." N-terminal epi-position " is epi-position or the antigenic determinant that is positioned at the N-terminal of A β peptide or comprises the N-terminal of A β peptide.Exemplary N-terminal epi-position is included in the 1-10 of A β or the residue in the 1-12 amino acids, 1-3,1-4,1-5,1-6,1-7,2-6,2-7,3-6 or the 3-7 position residue of preferred A β.Other exemplary N-terminal epi-position starts from the 1-3 position residue of A β, terminates in the 7-11 position residue of A β.Other exemplary N-terminal epi-position comprises 2-4,2-5,2-6,2-7 or the 2-8 position residue of A β, the 3-5 of A β, 3-6,3-7,3-8 or 3-9 position residue, or the 4-7 of A β, 4-8,4-9 or 4-10 position residue.

When certain antibody is called as when epi-position in specific residue such as the A β 3-7 combines, this means this antibody and the polypeptid specificity combination that contains specific residue (in this example promptly, A β 3-7).Such antibody not necessarily with A β 3-7 in each residue all contact.Each single amino acids in the A β 3-7 replaces or also appreciable impact binding affinity not necessarily of disappearance.In various embodiments, A β antibody is terminal specific.Term used herein " terminal specificity " is meant that antibody combines with the N-terminal or the C-terminal residue specificity of A β peptide, but is present in the longer A β kind apoplexy due to endogenous wind that comprises this residue or in APP the time, this antibody is these residues of nonrecognition but when identical residue.Preferred antibody has human IgG1's isotype.

The preferred anti-amyloid beta antibodies of passive immunotherapy comprises humanized anti-amyloid beta antibodies, for example, and humanization 3D6 antibody, humanization 12B4 antibody, or humanization 12A 11 antibody.

The antibody that is used for passive immunotherapy can provide by many kinds of technology, comprise technology described in US20040038304, US 20070020685, US 20060257396, US 20060160184, US 20060134098, US 20050255552, US 20050008625, US 20040132066, US 20040038317, US 20030198971 and the US 20030157579, all it is incorporated herein by reference in full with all purposes for it.

Antibody

I. the general characteristic of immunoglobulin

Known basic antibody structure unit comprises the tetramer of subunit.Each tetramer is made up of two pairs of identical polypeptide chains, and every pair has " gently " chain (about 25kDa) and " weight " chain (about 50-70kDa).The amino terminal of every chain partly comprises about 100 to 110 or the variable region of amino acids more, mainly is responsible for antigen recognition.The carboxyl terminal of every chain partly is defined as constant region, mainly is responsible for effector function.

Light chain is categorized as κ or λ.Heavy chain is categorized as γ, μ, α, δ or ε, and respectively the isotype of antibody is defined as IgG, IgM, IgA, IgD and IgE.In light chain and heavy chain, variable region and constant region by about 12 or more " J " district of amino acids engages, heavy chain comprises that also about amino acid whose more than 10 " D " distinguishes.(referring to, Fundamental Immunology, Ch.7 (" basic immunology " chapter 7, W.Paul writes, Raven Press, N.Y., second edition, 1989), it is incorporated herein by reference in full with all purposes).

The right variable region of each light/heavy chain forms antibody combining site.Therefore, complete antibody has two binding sites.Remove in difunctional or bi-specific antibody China and foreign countries, these two binding sites are identical.Chain all shows identical general structure: by three hypervariable regions, also claim the conservative relatively framework region (FR) that complementary determining region or CDRs engage.The CDRs of two chains that each is right aligns by framework region, thus can with the combining of specificity epitope.From the N-terminal to the C-terminal, light chain and heavy chain all comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domain.The amino acid whose distribution and the Kabat of each domain, Sequences of Proteins ofImmunological Interest (sequence of immunology important protein) (National Institutesof Health, Bethesda, MD, 1987 and 1991); Chothia and Lesk, J.Mol.Biol.196:901-917 (1987); Or Chothia etc., the definition unanimity of Nature 342:878-883 (1989).

Ii. non-human antibody's generation

The generation of non-human monoclonal antibodies, for example Mus, Cavia porcellus, pig, primate, rabbit or rat can be by for example finishing with A β immune animal.Also can use the immunogenic fragments of the anti-atopic antibody of the long peptide that comprises A β or A β or A β antibody.See Harlow and Lane, Antibody, A Laboratory Manual (antibody, laboratory manual) (CSHP NY, 1988) (being incorporated herein by reference) for all purposes.Such immunogen can be from natural source, the synthetic or recombinant expressed acquisition by peptide.Optional, immunogen can as mentioned belowly merge with carrier protein or otherwise compound back administration.Optional, immunogen can be with the adjuvant administration.Adjuvant that can use a few types as mentioned below.The preferred Freund's complete adjuvant of the laboratory animal immunity full adjuvant that toos many or too much for use then.Rabbit or Cavia porcellus are generally used for preparing polyclonal antibody.Mice is generally used for preparing monoclonal antibody.Screening and the bonded antibody of A β specificity.Optional, further screen the bonded antibody of given zone with A β.The back plant the combining of deletion mutant set that screening can be by measuring antibody and A β peptide, and definite which kind of deletion mutant and this antibodies finish.In conjunction with estimating by for example Western trace or ELISA.Show the epi-position that defines this antibody with the bonded minimal segment of antibody specificity.Perhaps, can measure epitope specificity by for examination antibody and reference antibody competition and the bonded competition analysis of A β.If for examination antibody and reference antibody competition, then they and identical epi-position or enough approaching, so that a kind of epi-position combination of the another kind of antibodies of combination interference of antibody.The preferred isotype of such antibody is the isotype that is equal to of mice isotype IgG2a or other species.Mice isotype IgG2a is the equivalents of people's isotype IgG1.

Iii. chimeric and humanized antibody

Chimeric and humanized antibody has and mice or same or analogous binding specificity of other non-human antibody and affinity that the parent material that makes up chimeric or humanized antibody is provided.Chimeric antibody is the antibody that makes up its light chain and heavy chain gene usually with the immunoglobulin gene section that belongs to different plant species by genetic engineering.For example, can constant (C) section from variable (V) section of the gene of mouse monoclonal antibody with the people such as IgG1 and IgG4 combine.People's isotype IgG1 is preferred.In certain methods, the isotype of antibody is a human IgG.Also can use IgM antibody in the certain methods.Therefore, typical chimeric antibody is by V or the C of antigen binding domain and people's antibody or the hybridization albumen that effector domain is formed of mouse antibodies.

Humanized antibody has basically from the variable region framework residue of people's antibody (being called receptor antibody) with basically from the mouse antibodies complementary determining region of (being called the donor immunity globulin).Referring to, Queen etc., Proc.Natl.Acad.Sci.USA 86:10029-10033 (1989), WO90/07861, US 5,693,762, and US 5,693,761, US 5,585,089, US 5,530,101 and Winter, US 5,225,539 (for all purposes, they are incorporated herein by reference separately in full).Constant region is as existing, also basically or fully from the human normal immunoglobulin.People's variable domain is selected from its framework sequence table usually and reveals people's antibody of height sequence homogeneity with the Mus variable region domain of the CDRs that derives.Heavy chain and variable region of light chain framework residue can be derived from identical or different human antibody sequences.Human antibody sequence can be the sequence of naturally occurring people's antibody, perhaps can be the consensus sequence of several people's antibody.See Carter etc., WO 92/22653.According to influencing, select some aminoacid of people variable region framework residue to replace to the CDR conformation and/or with antigen is bonded.These researchs that may influence are undertaken by simulation, replacement or the mutagenic effect investigating the amino acid whose characteristic on the ad-hoc location or rule of thumb observe specific amino acids.

For example, aminoacid should be commonly used to replace people's framework amino acid from the framework amino acid that is equal to of mouse antibodies not simultaneously between Mus variable region framework residue and selected people variable region framework residue, reasonably expects this aminoacid in the case:

(1) directly combine with antigen is non-covalent,

(2) be close to the CDR district,

(3) otherwise interact (for example, in about 6A in CDR district) with the CDR district, or

(4) participate in the VL-VH interface.

Other replaces the candidate is human normal immunoglobulin's rare receptor people framework amino acid on this position.These aminoacid can be used for from the equivalent site of mice donor antibody or from the aminoacid replacement of more typical human normal immunoglobulin's equivalent site.Other replaces the candidate is human normal immunoglobulin's rare receptor people framework amino acid on this position.The variable region framework of Humanized immunoglobulin shows usually and the consensus sequence of people variable region framework sequence or these sequences has at least 85% sequence homogeneity.

Iv. people's antibody

People's antibody of anti-A β provides by following multiple technologies.Some antibody is by competitive binding experiment or otherwise select, having the epitope specificity identical with specific mouse antibodies, as one of mouse monoclonal antibody as described in the embodiment XI.People's antibody also can be by only using A β fragment as immunogen, and/or the antibody of the deletion mutant set by screening A β screens specific epitope specificity.People's antibody preferably has human IgG1's isotype specificity.

(1) trioma method

Oestberg etc., Hybridoma 2:361-367 (1983); Oestberg, United States Patent (USP) 4,634,664; With Engleman etc., United States Patent (USP) 4,634,666 (for all purposes, they are incorporated herein by reference separately in full) have been described basic skills and have been used for the exemplary cell fusion companion SPAZ-4 of this method.The antibody producing cell system that obtains by this method is called as trioma because it passes from three kinds of cells---two kinds of mankind with a kind of mice.At first, mouse myeloma cell line and human B lymphocyte are merged, the xenogenesis hybrid cell of not produced antibody is as the SPAZ-4 cell line of the same Oestberg description.Heterogenous cell merges with the human B lymphocyte of immunity then, obtains producing antibody trioma cell line.Found that trioma more stably produces antibody than preparation from the common hybridoma of people's cell.

The bone-marrow-derived lymphocyte of immunity is available from blood, spleen, lymph node or the bone marrow of people's donor.The antibody of anti-if desired specific antigen or epi-position preferably uses its antigen or epi-position to carry out immunity.Immunity can be that body is interior or external.For immunity in the body, usually from A β, its fragment, contain the philtrum separation B cell of the anti-atopic antibody immunity of A β or segmental bigger polypeptide or A β antibody.In certain methods, the B cell separation is from the same patient who finally is applied Antybody therapy.For external immunity, bone-marrow-derived lymphocyte in the culture medium such as RPMI-1640 (seeing Engleman, the same) of replenishing 10% human plasma, is exposed to antigen 7-14 days time usually.

The bone-marrow-derived lymphocyte of immunity merges by known method and xenogenesis hybrid cell such as SPAZ-4.For example, cell is that the Polyethylene Glycol of 1000-4000 is handled about 5-10min at about 37 ℃ with the molecular weight of 40-50%.From integrative mixture, isolate cell, the crossbred of wanting (for example, HAT or AH) is had optionally and breeds in the culture medium.The clone that secretion has the antibody of required binding specificity identifies in conjunction with A β or its segmental ability by analyzing the trioma culture medium.The trioma that generation has the specific people's antibody of expection carries out sub-clone by limiting dilution technique, carries out In vitro culture then in culture medium.Test gained trioma cell line then in conjunction with A β or its segmental ability.

Although trioma is stable in heredity, its level that produces antibody is very not high.Expression can be by going into the antibody cloning of trioma one or more expression vectors, and this carrier is transformed in the mammal, antibacterial of standard or the yeast cells system increases.

(2) transgenic nonhuman mammal

People's antibody of anti-A β also can be produced by the genetically modified non-human transgenic mammal of the section with the human immunoglobulin gene's seat of encoding at least.Usually, the endogenous immunoglobulin genes seat of such transgene mammal is deactivation on function.The section of preferred this human immunoglobulin gene's seat comprises the not retracing sequence of heavy chain and light chain component.The deactivation of endogenous immunoglobulin genes and the introducing of exogenous immunoglobulin gene can realize by directed homologous recombination or introducing YAC chromosome.Can on function, reset the immunoglobulin components sequence by the transgene mammal that this process produces, and the repertoire of the various isotype antibodies of expressing human immunoglobulin gene coding, and do not express endogenous immunoglobulin genes.Having the mammiferous generation of these characteristics and characteristic has a detailed description in for example following: Lonberg etc., and WO93/1222, US 5,877,397, US 5,874, and 299, US 5,814,318, and US 5,789,650, US 5,770,429, US 5,661, and 016, US 5,633,425, and US 5,625,126, US 5,569,825, US 5,545, and 806, Nature 148,1547-1553 (1994), and NatureBiotechnology 14,826 (1996), Kucherlapati, WO 91/10741 (for all purposes, it is incorporated herein by reference separately in full).Transgenic mice is especially suitable.Anti-amyloid beta antibodies is by obtaining with A β or the immunity of its fragment render transgenic non-human mammal, as described in the same Lonberg or Kucherlapati.Monoclonal antibody is for example passed through, and will merge with the myeloma cell line that suits from these mammiferous B cells with conventional Kohler-Milstein technology to prepare.People's polyclonal antibody can also be provided as the serum form with the people of immunogenic agents immunity.Optional, such polyclonal antibody can use A β or other 4 amyloid as affinity reagent, concentrates by affinity purification.

(3) phage display method

Another method that obtains people's anti-amyloid beta antibodies is according to Huse etc., the described general scheme of Science246:1275-1281 (1989), screening DNA library from human B cell.Described in the trioma method, such B cell can available from through A β fragment, contain the people of A β or segmental longer polypeptide or anti-atopic antibody immunity.Optional, such B cell is available from the patient who finally accepts Antybody therapy.Select and A β or the bonded antibody of its fragment.Sequence of these antibody of clones coding (or binding fragment) and amplification then.Scheme and display technique of bacteriophage that Huse describes are combined, can be more effective.Referring to, for example, Dower etc., WO 91/17271 and McCafferty etc., WO 92/01047, and US 5,877, and 218, US 5,871,907, US5, and 858,657, US 5,837,242, US 5,733, and 743 and US 5,565,332 (for all purposes, it is incorporated herein by reference separately in full).In these methods, produced wherein the member shows the phage library of different antibodies at its outer surface.Antibody is shown as Fv or Fab fragment usually.By to A β peptide or its segmental affine enrichment, select to show to have the phage of required specific antibody.

In the variant of phage display method, can produce people's antibody of binding specificity with selected murine antibody.See Winter, WO 92/20791.In the method, the heavy chain or the variable region of light chain of selected murine antibody are used as parent material.For example variable region of light chain is as parent material if select, and then in the phage library of Gou Jianing, its member shows identical variable region of light chain (that is Mus parent material) and different variable region of heavy chaines.Variable region of heavy chain is available from resetting people's variable region of heavy chain library.Selection shows that to A β strong specificity is in conjunction with (for example, at least 10 ⁸, preferably at least 10 ⁹M ^-1) phage.People's variable region of heavy chain of this phage plays the effect of the parent material that makes up another phage library then.In this library, variable region of heavy chain that each phage display is identical (that is the district that from first display libraries, differentiates) and different variable region of light chains.Variable region of light chain is available from people's variable region of light chain library of resetting.Once more, select A β is demonstrated the bonded phage of strong specificity.These phage displays are the variable region of people's anti-amyloid beta antibodies completely.These antibody have and the same or analogous epitope specificity of Mus parent material usually.

(4) nano antibody (NANOBODY) method

Anti-amyloid beta antibodies can also pass through Nanobody ^TMMethod (Ablynx N.V.) produces.Nano antibody is a human cytokines deutero-by antibody, that have naturally occurring heavy chain antibody characteristic.Nano antibody can play single, relatively little functional antigen integrated structure unit, domain or proteic effect.Nanobody ^TMThe initial exploitation of technology is after finding that camellid (camel and yamma) has the global function antibody of disappearance light chain.These heavy chain antibodies contain single variable domain (VHH) and two constant domains (CH2 and CH3).VHH is used for they are distinguished mutually with the heavy chain variable domain (being called " VH territory ") in being present in 4 conventional chain antibodies.Clone and isolating VHH territory are the stable polypeptide that comprises whole antigen binding capacities of original heavy chain antibody.VHH territory and nano antibody also can be engineered to multivalent and polyspecific form.Have with the nano antibody of the aminoacid sequence amino acid sequence corresponding in naturally occurring VHH territory can humanization, that is, by the one or more amino acid residues in the aminoacid sequence of naturally occurring VHH sequence (with framework sequence especially) are substituted with the one or more amino acid residues on the relevant position in the VH territory that is present in conventional people's 4 chain antibodies.Details sees, for example, US20050130266, US 20040253638, WO/2006/040153, US 20050214857, WO/2006/079372, or WO/2006/122825, for all purposes, it is incorporated herein by reference separately.

V. the selection of constant region

The heavy chain of chimeric, humanization or people's antibody can be connected with at least a portion of human constant region with variable region of light chain.The selection of constant region depends in part on whether antibody dependent complement and/or the cell-mediated toxicity of wanting.For example, isotype IgG1 and IgG3 have complement activity, and isotype IgG2 and IgG4 then do not have.The selection of isotype can also influence antibody and enter brain.People's isotype IgG1 is preferred.Constant region of light chain can be λ or κ.Antibody can be expressed as the tetramer that contains two light chains and two heavy chains, is expressed as separately heavy chain, light chain, is expressed as Fab, Fab ' F (ab ') 2 and Fv, perhaps is expressed as the single-chain antibody that wherein heavy chain and light chain variable territory are connected by spacer.

Vi. the expression of recombinant antibodies

Chimeric, humanization and people's antibody are usually by recombinant expressed generation.The recombination of polynucleotide construct generally includes the expression control sequenc that is operably connected with the coded sequence of antibody chain, and it comprises natural relevant or allogenic promoter region.Preferably, expression control sequenc be can transform or the carrier of transfection eukaryotic host cell in the eukaryotic promoter system.In case this carrier enters suitable host, the host just remains under the state that is suitable for high level expression nucleotide sequence and collection and purification fork reagin.

These expression vectors can duplicate by the integrated part as episome or host chromosome DNA in host's organism usually.Usually, expression vector contains selected marker, and for example, ampicillin resistance or hygromycin resistance are so that detect the DNA sequence cell transformed that these are wanted.

Escherichia coli are a kind of prokaryotic hosts that are particularly useful for cloning DNA sequence of the present invention.Microorganism as yeast, also can be used for expressing.Saccharomycodes (Saccharomyces) is preferred yeast host, and it has the appropriate carrier that contains required expression control sequenc, origin of replication, terminator sequence etc.Typical promoter comprises glycerol 3-phosphate acid kinase and other glycolytic ferment.The induction type Yeast promoter is particularly including the promoter from the enzyme of alcoholdehydrogenase, different cell pigment (isocytochrome) C and responsible maltose and galactose utilization.

Mammalian cell is the preferred host who expresses immunoglobulin or its segmental coding nucleotide section.See Winnacker, From Genes to Clones (from gene to clone), (VCHPublishers, NY, 1987).It is to comprise Chinese hamster ovary celI system, various COS cell line, HeLa cell, L cell, HEKC and myeloma cell line that the many suitable host cells that can secrete complete heterologous protein have been developed in this area.Preferably, cell is inhuman.The expression vector of these cells can comprise expression control sequenc, as origin of replication, promoter, enhancer (Queen etc., Immunol.Rev.89:49 (1986)), machining information site with necessity, as ribosome binding site, RNA splice site, polyadenylation site, and transcription terminator.Preferred expression control sequenc is the promoter derived from endogenous gene, cytomegalovirus, SV40, adenovirus, bovine papilloma virus etc.See Co etc., J.Immunol.148:1149 (1992).

Perhaps, the antibody of coded sequence can be incorporated in the transgenic, to be introduced into the genome of transgenic animal, in the milk of these transgenic animal, express subsequently (referring to, for example, US 5,741,957, and US 5,304,489, and US 5,849, and 992).Suitable transgenic comprise with from the promoter of mammary gland specific gene such as casein or beta lactoglobulin and the light chain that enhancer is operably connected and/or the coded sequence of heavy chain.

The carrier that contains the target DNA section can change in the host cell by known method according to the type of cell host.For example, the calcium chloride transfection is usually used in prokaryotic cell, and calcium phosphate processing, electroporation, lipofection, particle gun (biolistics) or can be used for other cell host based on the transfection of virus.Other method that is used for transformed mammalian cell comprises utilization polybrene, protoplasm fusion, liposome, electroporation and microinjection (generally see, Sambrook etc., the same).For producing transgenic animal, the transgenic microinjection can be gone into the oocyte of fertilization, perhaps can be integrated in the genome of embryonic stem cell, and the nucleus of these cells is changed in the enucleation oocyte.

Antibody comprises (generally seeing Scopes, Protein Purification (protein purification) (Springer-Verlag, NY, 1982)) such as HPLC purification, column chromatography, gel electrophoresiss in case expression just can be carried out purification according to the standardization program of this area.

Chimeric or the humanization form of 3D6 or its is preferred antibody (seeing U.S. Patent Publication No.20030165496A1, U.S. Patent Publication No.20040087777A1, international patent publications No.WO 02/46237A3 and international patent publications No.WO 04/080419A2).The description of 3D6 be also shown in, for example, international patent publications No.WO 02/088306A2 and international patent publications No.WO02/088307A2.Other 3D6 antibody is at U.S. Patent application No.11/303,478 and international patent application No.PCT/US05/45614 in describe.3D6 is and is arranged in people's beta-amyloyd peptide N-terminal epi-position, is specially the bonded monoclonal antibody of 1-5 position residue specificity (mAb).Clause according to budapest treaty (Budapest Treaty), the cell line (RB963D6.32.2.4) that produces the 3D6 monoclonal antibody is deposited in American type culture collection (American Type Culture Collection on April 8th, 2003, ATCC), Manassas, VA 20108, USA, preserving number are PTA-5130.

Ba Biniu pearl monoclonal antibody (bapineuzumab) is meant a kind of humanization 3D6 antibody, its light chain that comprises has ripe variable region and the heavy chain that aminoacid sequence is designated as SEQ ID NO:1 and has the ripe variable region that aminoacid sequence is designated as SEQ ID NO:2, shows below.

Humanization 3D6 variable region of light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro

Val?Thr?Pro?Gly?Glu?Pro?Ala?Ser?Ile?Ser?Cys?Lys?Ser?Ser?Gln?Ser

Leu?Leu?Asp?Ser?Asp?Gly?Lys?Thr?Tyr?Leu?Asn?Trp?Leu?Leu?Gln?Lys

Pro?Gly?Gln?Ser?Pro?Gln?Arg?Leu?Ile?Tyr?Leu?Val?Ser?Lys?Leu?Asp

Ser?Gly?Val?Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe

Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr

Cys?Trp?Gln?Gly?Thr?His?Phe?Pro?Arg?Thr?Phe?Gly?Gln?Gly?Thr?Lys

Val?Glu?Ile?Lys(SEQ?ID?NO：1)

Humanization 3D6 variable region of heavy chain

Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln

Pro?Gly?Gly?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe

Ser?Asn?Tyr?Gly?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu

Glu?Trp?Val?Ala?Ser?Ile?Arg?Ser?Gly?Gly?Gly?Arg?Thr?Tyr?Tyr?Ser

Asp?Asn?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn

Thr?Leu?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val

Tyr?Tyr?Cys?Val?Arg?Tyr?Asp?His?Tyr?Ser?Gly?Ser?Ser?Asp?Tyr?Trp

Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser(SEQ?ID?NO：2)

Ba Biniu pearl monoclonal antibody (bapineuzumab) is called as AAB-001.

Second kind of version humanization 3D6 antibody, its light chain that comprises has ripe variable region and the heavy chain that aminoacid sequence is designated as SEQ ID NO:3 and has the ripe variable region that aminoacid sequence is designated as SEQ ID NO:4, shows below.

Humanization 3D6 variable region of light chain

Tyr?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro

Val?Thr?Pro?Gly?Glu?Pro?Ala?SerIle?Ser?Cys?Lys?Ser?Ser?Gln?Ser

Leu?Leu?Asp?Ser?Asp?Gly?Lys?Thr?Tyr?Leu?Asn?Trp?Leu?Leu?Gln?Lys

Pro?Gly?Gln?Ser?Pro?Gln?Arg?Leu?Ile?Tyr?Leu?Val?Ser?Lys?Leu?Asp

Ser?Gly?Val?Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe

Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr

Cys?Trp?Gln?Gly?Thr?His?Phe?Pro?Arg?Thr?Phe?Gly?Gln?Gly?Thr?Lys

Val?Glu?Ile?Lys(SEQ?ID?NO：3)

Humanization 3D6 variable region of heavy chain

Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln

Pro?Gly?Gly?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe

Ser?Asn?Tyr?Gly?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu

Glu?Trp?Val?Ala?Ser?Ile?Arg?Ser?Gly?Gly?Gly?Arg?Thr?Tyr?Tyr?Ser

Asp?Asn?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala?Lys?Asn

Ser?Leu?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Leu

Tyr?Tyr?Cys?Val?Arg?Tyr?Asp?His?Tyr?Ser?Gly?Ser?Ser?Asp?Tyr?Trp

Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser(SEQ?ID?NO：4)

The third version humanization 3D6 antibody comprise aminoacid sequence and be designated as the light chain of SEQID NO:5 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:6, in US 2005/0090649 A1 that announced on April 28th, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 3D6 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Leu?Gly

Gln?Pro?Ala?Ser?Ile?Ser?Cys?Lys?Ser?Ser?Gln?Ser?Leu?Leu?Asp?Ser

Asp?Gly?Lys?Thr?Tyr?Leu?Asn?Trp?Leu?Gln?Gln?Arg?Pro?Gly?Gln?Ser

Pro?Arg?Arg?Leu?Ile?Tyr?Leu?Val?Ser?Lys?Leu?Asp?Ser?Gly?Val?Pro

Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile

Ser?Arg?Val?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Trp?Gln?Gly

Thr?His?Phe?Pro?Arg?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys

Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu

Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe

Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln

Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser

Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu

Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser

Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys(SEQ?ID?NO：5)

Humanization 3D6 heavy chain

Gln?Val?Gln?Leu?Val?Gln?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly

Ser?Leu?Arg?Leu?Ser?Cys?Ala?Gly?Ser?Gly?Phe?Thr?Phe?Ser?Asn?Tyr

Gly?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val

Ala?Ser?Ile?Arg?Ser?Gly?Gly?Gly?Arg?Thr?Tyr?Tyr?Ser?Asp?Asn?Val

Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Glu?Asn?Ala?Lys?Asn?Ser?Leu?Tyr

Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

Val?Arg?Tyr?Asp?His?Tyr?Ser?Gly?Ser?Ser?Asp?Tyr?Trp?Gly?Gln?Gly

Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe

Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu

Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Gln?Pro?Val?Thr?Val?Ser?Trp

Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu

Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser

Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro

Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys

Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Gln?Leu?Leu?Gly?Gly?Pro

Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser

Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp

Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn

Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val

Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu

Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys

Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr

Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr

Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu

Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu

Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys

Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu

Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly

Lys(SEQ?ID?NO：6)

Chimeric or humanization of 12A11 or its or nano antibody form are preferred antibody.12A11 antibody or its variant are described in U.S. Patent Publication No.20050118651, U.S. Patent Publication No.20060198851, international patent publications No.WO 04/108895 and international patent publications No.WO 06/066089, for all purposes, it all is incorporated herein by reference in full.12A11 is with the N-terminal epi-position that is positioned at people's beta-amyloyd peptide, is specially the bonded mAb of 3-7 position residue specificity.The cell that produces the 12A11 monoclonal antibody lies in December in 2005 and was deposited in ATCC (American type culture collection, 10801 University Boulevard, Manassas, VA 20110-2209) on the 13rd, and the ATCC accession number is PTA-7271.

The humanization 12A11 antibody of first kind of version, it comprises aminoacid sequence and is designated as the light chain of SEQ ID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:8 (version 1), in the US 20050118651A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 1)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?LeuAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Val?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?Thr?Thr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：8)

The humanization 12A11 antibody of second kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:9 (version 2), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 2)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Thr?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?ValAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Phe?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Leu?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?No：9)

The third version humanization 12A11 antibody comprise aminoacid sequence and be designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:10 (version 2 .1), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 2 .1)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Thr?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?ValAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Phe?Thr?Ile?Ser?Lys?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：10)

The 4th kind of version humanization 12A11 antibody comprise aminoacid sequence and be designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:11 (version 3), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?GlyGln?Ser?Pro?Gln?Leu?Leu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 3)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Thr?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?ValAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：11)

The humanization 12A11 antibody of the 5th kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:12 (edition 4 .1), in US 20050118651 A1 that announced on June 2nd, 2005, describe, all purposes of coming in and going out, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (edition 4 .1)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Thr?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?LeuAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Val?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：12)

The humanization 12A11 antibody of the 6th kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:13 (edition 4 .2), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (edition 4 .2)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?ValAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Val?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?Thr?Thr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：13)

The humanization 12A11 antibody of the 7th kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:14 (edition 4 .3), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (edition 4 .3)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?LeuAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Phe?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Val?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：14)

The humanization 12A11 antibody of the 8th kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:15 (edition 4 .4), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (edition 4 .4)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?LeuAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Leu?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：15)

The humanization 12A11 antibody of the 9th kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:16 (version 5.1), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?Leu?Leu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 5.1)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Thr?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?ValAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Val?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：16)

The humanization 12A11 antibody of the tenth kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:17 (version 5.2), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 5.2)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Thr?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?LeuAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Phe?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Val?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：17)

The humanization 12A11 antibody of the 11 kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:18 (version 5.3), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 5.3)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Thr?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?LeuAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Leu?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?Thr?Thr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser?Val(SEQ?ID?NO：18)

The humanization 12A11 antibody of the 12 kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:19 (version 5.4), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 5.4)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?ValAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Phe?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Val?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser?Val(SEQ?ID?NO：19)

The humanization 12A11 antibody of the 13 kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:20 (version 5.5), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 5.5)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?ValAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Leu?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：20)

The humanization 12A11 antibody of the 14 kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:21 (version 5.6), in the US 20050118651A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?Leu?Leu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 5.6)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?LeuAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Phe?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Leu?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：21)

The humanization 12A11 antibody of the 15 kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:22 (version 6.1), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 6.1)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Thr?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?ValAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Phe?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Val?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：22)

The humanization 12A11 antibody of the 16 kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:23 (version 6.2), in the US 20050118651A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 6.2)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Thr?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?ValAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Leu?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?Thr?Thr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：23)

The humanization 12A11 antibody of the 17 kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:24 (version 6.3), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 6.3)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Thr?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?LeuAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Phe?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Leu?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?sp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?SerSer(SEQ?ID?NO：24)

The humanization 12A11 antibody of the 18 kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:25 (version 6.4), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 6.4)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?ValAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Phe?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Leu?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：25)

The humanization 12A11 antibody of the 19 kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:26 (version 7), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein by reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?Leu?Leu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 7)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Thr?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?LeuAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Lys?Asn?Thr?Val?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：26)

The humanization 12A11 antibody of the 20 kind of version comprises aminoacid sequence and is designated as the light chain of SEQID NO:7 and the heavy chain that aminoacid sequence is designated as SEQ ID NO:27 (version 8), in US 20050118651 A1 that announced on June 2nd, 2005, describe, for all purposes, this application is incorporated herein as reference.

Humanization 12A11 light chain

Asp?Val?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro?Val?Thr?Pro?GlyGlu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser?Ile?Val?His?Ser?Asn?GlyAsn?Thr?Tyr?Leu?Glu?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?LeuLeu?Ile?Tyr?Lys?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro?Asp?Arg?Phe?SerGly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?AlaGlu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Phe?Gln?Ser?Ser?His?Val?Pro?Leu?ThrPhe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys(SEQ?ID?NO：7)

Humanization 12A11 heavy chain (version 8)

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?ArgSer?Leu?Arg?Leu?Ser?Cys?Ala?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser?GlyMet?Ser?Val?Gly?Trp?Ile?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?LeuAla?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?SerArg?Leu?Thr?Ile?Ser?Lys?Asp?Asn?Ser?Lys?Asn?Thr?Val?Tyr?Leu?Gln?MetAsn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?ThrThr?Thr?Ala?Asp?Tyr?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?ValSer?Ser(SEQ?ID?NO：8)

Above-mentioned any antibody can produce with different isotypes or mutant isotype, with the degree of control with different Fc receptors bind.Lack the Fc district antibody (for example, Fab fragment) can not with the Fc receptors bind.The selection of isotype also influences and the combining of Fc receptor.After measured various human IgG isotypes separately to the affinity (seeing Ravetch and Kinet, Annu.Rev.Immunol.9,457 (1991)) of three kinds of Fc γ receptor FcRI, FcRII and FcRIII.FcRI is and the bonded high-affinity receptor of the IgG of monomeric form, back two kinds be only with the bonded low-affinity receptor of the IgG of polymer form.Generally speaking, IgG1 and IgG3 have remarkable in active to all three kinds of receptors, and IgG4 is only to FcRI, and IgG2 only is called as a type of IIaLR to FcRII and has remarkable in active (seeing Parren etc., J.Immunol.148,695 (1992)).Therefore, when expectation and Fc γ receptors bind are strong, often select people's isotype IgG1, and IgG2 is often selected in more weak combination.

The hinge bonding pad of all isotypes is close to or has reduced Fc γ receptor, the especially affinity of Fc γ RI receptor near the sudden change on the site (for example, 234,235,236 and/or 237 residues are substituted by another residue).Optional, 234,236 and/or 237 are replaced by alanine, 235 replaced by glutamine (referring to, for example, US 5,624,821).236 disappearances in human IgG2's isotype.Human IgG2's 234,235 and 237 exemplary aminoacid section is ala ala gly, val ala ala, ala ala ala, val glu ala and ala glu ala.For people's isotype IgG1, preferred mutant combination is L234A, L235A and G237A.Especially preferred antibody is the Ba Biniu pearl monoclonal antibody (bapineuzumab) with these three kinds of sudden changes in people's isotype IgG and Fc district.Other reduce with Fc γ receptors bind be substituted with E233P sudden change (especially in mice IgG1) and D265A.

The aminoacid of constant region is by being numbered (seeing Cunningham etc., J.Biol.Chem., 9,3161 (1970)) with people's antibody EU comparison.That is, the heavy chain of antibody and heavy chain and the light chain of light chain and EU are compared, make amino acid sequence identity maximization, and with each aminoacid in this antibody distribute with EU in the identical numbering of corresponding aminoacid.The EU numbering system be conventional (referring to, Kabat etc., Sequences of Protein of Immunological Interest (immune important protein sequence), NIH publication No. 91-3242, US Department of Health andHuman Services (U.S. HHS) (1991)).

Antibody to the affinity of complement component Clq can by make heavy chain 318,320 with 322 amino acids residues at least one be mutated into residue and change with different side chains.For change, for example reduce or eliminate, with bonded other suitable change of the specific C lq of antibody, comprise that in 318 (Glu), 320 (Lys) and 322 (Lys) the residues any one becomes Ala.Clq can eliminate by in described three specific residues any one substituted with the residue that has inappropriate functionality on its side chain in conjunction with activity.Needn't only eliminate the Clq combination with Ala substitutional ion residue.In order to eliminate the Clq combination, with nonionic residue such as Gly, Ile, Leu or the Val of other alkyl replacement, or aromatic series non-polar residue such as Phe, Tyr, Trp and Pro, any one that substitutes in three residues also is possible.In addition, for eliminating Clq, substitute 320 and 322 residues with polarity nonionic residue such as Ser, Thr, Cys and Met, rather than 318 residues also are possible in conjunction with activity.Substituting 318 (Glu) position residues with polar residues can change but not eliminate Clq in conjunction with activity.Substitute the removal that 297 (Asn) position residues have caused lytic activity with Ala, and to the affinity of Clq only slightly descend (weakening three times approximately).This change has destroyed the existence of glycosylation site and the necessary carbohydrate of complement activation.Any other replacement on this site also destroys glycosylation site.

III. be suitable for the patient of (CAA) therapeutic scheme

Cerebral amyloid angiopathy is also referred to as has a liking for Congo red angiopathy or cerebrovascular amyloidosis.This is the little angiopathy of a kind of brain, and wherein, the amyloid beta deposition thing of blood vessel wall can cause apoplexy, cerebral hemorrhage, white matter ischemia or dementia.The similar starch of amyloid is deposited in the tissue in the course of disease of some chronic disease.

CAA can attack the patient more than 45 years old, but is most commonly in the over-65s patient, and becomes more common with age.Masculinity and femininity is ill to be equated.In some cases, CAA is sporadic, but its also can with autosomal dominant inherited disease (a kind of mode of inheritance, wherein only need there be a copy of the encoding gene of this disease in the expression of disease; Either party suffers from this when sick father and mother, and it is 50% that children inherit this sick probability) heredity.CAA is responsible to the cerebral hemorrhage of 5-20% and high cerebral lobar hemorrhage to a leaf that is confined to brain of 30%.Even there was not other performance of cerebral hemorrhage, apoplexy or this disease when alive, but when postmortem, can find CAA above 1/3rd people more than 60 years old.In Alzheimer's disease, CAA is than more common and can exist in greater than 80% 60 above Alzheimer's disease patients in the general population.

The reason of cerebrovascular amyloid deposition is still not clear among the sporadic CAA.Accumulation that in heritability CAA, genetic defect on No. 21 chromosomes, has allowed amyloid usually---a kind of albumen that is made of the fibriilar unit of βZhe Die sheet by name---.Fibril has forms one trend, makes amyloid not dissipate, and accumulation is got up in the cerebrovascular wall.A kind of form of amyloid fibrils protein subunit is an amyloid beta protein.

Amyloid deposition can destroy endotheliocyte or smooth muscle cell, or endotheliocyte and smooth muscle cell, perhaps causes vessel wall inflammation, and can cause that blood vessel is easier to break.When the capillary vessel that has amyloid deposition becomes more and more heavier, more and more crisp, and when therefore more likely because of the blood pressure of little damage or fluctuation explosion taking place, intracerebral hemorrhage can also take place.When tensile wall becomes thinner and be under the bigger pressure, may form aneurysm or blood vessel wall and expand, also may break.

The most common form of CAA is the distribute form relevant with aging.Such CAA causes usually may be at the cerebral lobar hemorrhage of the different leaves recurrence of brain.Frontal lobe (behind volume) and top (behind frontal lobe) are the most normal affected; Then less invasion and attack of temporal lobe (near temples) and occipital lobe (at the brain rear portion); And cerebellum (under occipital lobe) is seldom attacked.Among the sporadic CAA, about 10-50% hemorrhage relates to more than one cerebral lobe.

In CAA, the symptom of cerebral lobar hemorrhage is according to the cerebral lobe that is involved, and comprising that headache breaks out, and nervous symptoms such as weak, anesthesia, the visual field change or speech disorder; And level of consciousness descends (patient is difficult to wake up) nausea and vomiting.Sporadic CAA can with the irrelevant symptom of cerebral lobar hemorrhage.Petechial hemorrhage (involving the small hemorrhage of many little blood vessels) can cause the of short duration nervous symptoms of recurrent that is secondary to epilepsy or blood flow minimizing, perhaps can cause the rapid progressivity dementia (memory and other brain function are lost) that worsens rather than progressively worsen by distinct steps.CAA secondary hemorrhage patient above 40% suffers from dementia simultaneously.

Inherited genetic factors works in the CAA of some type and CAA relevant disease:

Holland's type (Dutch) Hereditary cerebral hemorrhage with amyloidosis (amyloid is accumulated in blood vessel): autosomal dominant relates to the gene mutation of amyloid precursor protein.Fallen ill in 40-60 year, often be positioned at cerebral hemorrhage, apoplexy and the dementia of top with headache.Patient over half dies from its first blood.It is unusual or blood is thinning that anticoagulant can take place Holland type CAA patient, and this causes more likely hemorrhage.

Flemish type Hereditary cerebral hemorrhage with amyloidosis: autosomal dominant, with the sudden change that relates to amyloid precursor protein.Symptom comprises cerebral hemorrhage or dementia.

The familial Alzheimer's disease: autosomal dominant accounts for all Alzheimer's disease cases 5-10% of (wherein nerve cell death causes the encephalopathy of progressive dementia).

Mongolism: cause by trisomy 21 (3 rather than 2 No. 21 chromosome copies), cause the amyloid precursor protein gene excessive.The mongolism infant has intelligence disability, and has cardiac problems.

Iceland's type Hereditary cerebral hemorrhage with amyloidosis: autosomal dominant, with the encoding gene sudden change of cysteine proteinase inhibitor C.Symptom often starts from 30-40 year, with dead in multiple cerebral hemorrhage, dementia, paralysis (weak) and the 10-20.Patient over half has a headache, and 1/4th epilepsy occurs.Different with the CAA of most of other form, most of hemorrhage ganglion basals that involve depths in the brain.(ganglion basal be brain cerebellum part organize the island).

Familial eye-pia arachnoid amyloidosis: autosomal dominant, genetic flaw the unknown sees Japan, Italy and North America family.Symptom can comprise dementia, ataxia (dystaxia), and spasm (extremity are stiff), apoplexy, epilepsy, peripheral neurophaty (disease of the nerve of influence domination extremity), migraine, the spinal cord problem, blind and deaf.Rare cerebral hemorrhage, because amyloid beta deposition is in the blood vessel of eye and meninges (tunicle of brain), rather than in the brain self.In the ill family of Italy, the patient can just be subjected to the invasion and attack of this disease as far back as 20-30 during year.

Britain's type familial amyloid sample becomes: autosomal dominant, genetic flaw the unknown is with progressive dementia, spasm and ataxia.Brain stem, spinal cord and cerebellum all show amyloid deposition, but do not take place hemorrhage usually.

In certain methods, suffer from CAA and do not have Alzheimer's disease or the patient of other disease symptoms is suitable for maybe inducing the medicament of A β antibody to treat with A β antibody.In other method, the patient of concurrent CAA and Alzheimer's disease or other disease is suitable for maybe inducing the medicament of A β antibody to treat with A β antibody.In other method, the patient who does not suffer from CAA and Alzheimer's disease and other any disease is suitable for maybe inducing the medicament of A β antibody to treat with A β antibody.

In the asymptomatic patient, treatment can start from any age (for example, 10,20 or 30 years old).Yet, there is no need usually the patient reach 40,50,60 or 70 years old before begin treatment.Treatment must be multiple dosing in a period of time usually.Treatment can be monitored replying of healing potion (antibody of A β or its fragment) by analyzing antibody or activated T cell or B cell in time.Weaken if reply, show and answer escalated dose.

Optional, whether the existence of symptom, sign or the risks and assumptions of definite disease before begin treatment.

IV.CAA patient's diagnosis and monitoring

The same with most of sacred diseases, usually draw diagnosis from the pattern of patient's medical history and careful inquiry family history and patient's incidence and symptom and neurologic examination.Brain computed tomography (CT) or NMR (Nuclear Magnetic Resonance)-imaging (MRI) can be identified cerebral lobar hemorrhage, apoplexy or petechial hemorrhage, and be very important in getting rid of arteriovenous malformotion, the cerebral tumor or other cause of bleeding.Angiography (the X ray examination of blood vessel and heart inside) does not have help to the diagnosis of CAA, but may be that the eliminating aneurysm is needed.Biopsy of brain (excision fritter cerebral tissue) can the indicating characteristic amyloid deposition.When diagnosis is uncertain, may need biopsy to get rid of the potential disease that will treat.Check unusual that the lumbar puncture of cerebrospinal fluid protein can indicating characteristic.

Must make a distinction with the cerebral hemorrhage of other type with hemorrhage CAA.In CAA, the hemorrhage leaf district that usually occurs in, often breaking enters brain and by in the intermembranous subarachnoid space, and takes place at night.In relevant hemorrhage of hypertension, the hemorrhage more depths that usually occurs in the brain, breaking enters in the chamber or chamber of depths in the brain, and takes place when day's activities.Other reason of cerebral hemorrhage has arteriovenous malformotion, wound, and aneurysm, hemorrhage in the cerebral tumor, vasculitis (inflammation of blood vessel) or hemorrhagic disease.The patient can monitor little hemorrhage and/or the removal by positron emission fault (PET) scanning monitoring blood vessel amyloid of brain by MRI.

The patient who is suitable for treating comprises the individuality that is in the CAA risk but does not show symptom, and the patient who is just showing symptom at present.

V. therapeutic scheme

In prophylactic use, to easy trouble CAA or be in patient's drug administration compositions or medicament in the CAA risk, scheme comprises that the dosage of described compositions or medicament and frequency are enough to eliminate or reduce risk, alleviate severity or postpone seizure of disease, the physiology, biochemistry, histology and/or the behavior symptom that comprise disease, the complication that presents in the disease progression process and middle pathology phenotype.In therapeutic is used, the patient who suffers from or suffered from such disease to suspection uses compositions or medicament, scheme comprises the dosage of described compositions and the symptom (physiology that frequency is enough to cure or contain to small part disease, biochemistry, histology and/or behavior), comprise the complication and the middle pathology phenotype that present in the disease progression.Finish therapeutic or preventative-therapeutic appropriate amount and be defined as effectively treatment or preventive dose.Finish therapeutic or preventative-therapeutic suitable amount and the combination of administration frequency and be defined as effectively treatment or prevention scheme.In prevention and therapeutic scheme, the common administration of medicament is dosage for several times, until obtaining sufficient immunne response.Usually, the monitoring immunne response, and when immunne response begins to weaken repeat administration.

The effective dose of the above-mentioned disease of combination treatment of the present invention changes according to many different factors, and physiological status, the patient who comprises administering mode, target site, patient is that people or animal, the other medicines of using and treatment are preventative or curative.Usually, the patient is the people, but also can treat non-human mammal, comprises transgene mammal.Therapeutic dose needs to increase gradually, makes safety and effectiveness the best.Whether immunogenic measuring certainly also uses in adjuvant, needs more high dose during no adjuvant.When people's administration, immunogenic dosage changes between 1-500 μ g/ patient sometimes, is more typically in change between 5-500 μ g/ injection.Usually, everyone injects and uses about 10,20,50 or 100 μ g.Immunogenic quality also depends on the interior immunogenicity epi-position of immunogen and the mass ratio of immunogen oeverall quality.Usually, use 10 for the immunogen of Gamma Magnitude ^-3-10 ^-5The immunogenicity epi-position of μ mol.The selection of inject time can be from once a day to once and significantly different by per ten years once a year.Give the settled date giving any of immunogen dosage, if also use adjuvant, then dosage is greater than 1 μ g/ patient, and usually greater than 10 μ g/ patients, during no adjuvant, and greater than 10 μ g/ patients, and usually greater than 100 μ g/ patients.Typical scheme is by first immunity, form as the interval booster injection in 6 weeks by interval then.Another program by first immunity, then 1,2 and 12 months after booster injection form.Another program needs injection in per 2 months throughout one's life once.Perhaps, booster injection can be carried out at random according to shown in the monitoring of immunne response.

For carrying out passive immunity with antibody, dosage is generally 0.01-5mg/kg host's body weight.Especially, dosage range is about 0.5 to less than 5mg/kg, is more typically 0.5-3mg/kg host's body weight.For example, dosage can perhaps in the scope of 0.5-1.5mg/kg, be preferably 1.5mg/kg at least less than 5mg/kg body weight or 1.5mg/kg body weight.Object can every day, every other day, use these dosage once in a week or according to any timetable that other is determined by empirical analysis.Exemplary treatment need be in the long period, for example at least 6 months, and multiple dose administration.Other exemplary therapeutic scheme need per 2 weeks once or every month once or every 3-6 month single administration.

Exemplary passive administration time table comprises per 13 all 1.5-3mg/kg or 1.5mg/kg.The common multiple dosing of medicament of the present invention.Interval between single dose can be weekly, every month, per 13 weeks or annual.Interval can also be irregular according to shown in the measurement of the A β antibody horizontal in the blood samples of patients.

In certain methods, adjust dosage to reach the plasma antibody concentration of 1-1000 μ g/ml, in certain methods, reach 25-300 μ g/ml.Perhaps, antibody can be used as the slow releasing preparation administration, and in this case, the administration frequency that needs is lower.Dosage and frequency changed according to the half-life of antibody in the patient.Generally speaking, people's antibody demonstrates the longest half-life, is thereafter humanized antibody, chimeric antibody and non-human antibody.

The preferred version of using the specific antibody of A β N-terminal reaches the average serum concentration 1-15 μ g/ml of institute's administration of antibodies in the patient.Serum-concentration can be determined by actual measurement, perhaps (for example from the standard pharmacokinetics, the WinNonline4.0.1 version (Pharsight Corporation, Cary, USA)) according to predicting dosage, administration frequency, route of administration and the antibody half life of antibody.Average antibody concentration in the serum is preferably in the scope of 1-10,1-5 or 2-4 μ g/ml.

For intravenous administration, every month once to per season once (preferred per 13 weeks once) application dosage be the antibody of 0.1-5mg/kg.For per season single administration, dosage is preferably in the scope of 0.5-3,0.5-2 or 0.5-1.5mg/kg.The preferred dose of every month intravenous administration antibody once is at the antibody of 0.1-1.0mg/kg, or in the antibody scope of preferred 0.5-1.0mg/kg.

For more frequent administration, for example, weekly to every month single administration, subcutaneous administration is preferred.The dosage that is used for subcutaneous administration is usually in the scope of 0.1-0.6mg/kg or 0.01-0.35mg/kg, preferred 0.05-0.25mg/kg.For once in a week or administration biweekly whenever, dosage is preferably in the scope of 0.015-0.2mg/kg or 0.05-0.15mg/kg.For weekly administration, dosage is preferably 0.05-0.07mg/kg, for example, and 0.06mg/kg.For administration biweekly whenever, dosage is preferably 0.1-0.15mg/kg.For every month single administration, dosage was preferably 0.1-0.3mg/kg or 2mg/kg.Single administration comprised according to calendar month or lunar month (that is, whenever all around once) administration in every month.

Therapeutic scheme is continuous usually, so that the average serum concentration of above-mentioned antibody kept 6 months or 1 year at least, and all one's life sometimes.Serum-concentration can be measured any time in therapeutic process, if mean concentration falls into below the target zone, then increases dosage and/or frequency, if perhaps mean concentration falls into more than the target zone, then reduces dosage and/or frequency.

Although it is useful for the dosage of determining individual patient or optimization dosage to measure the best plasma concentration of antibody, but in fact, in case determined the effective dose scheme according to the mg/kg of administration or mg and frequency, many other patients just can use identical dosage, and need not detailed calculated or measure patient's titre.Therefore, no matter whether measure or predict concrete patient's titre, above-mentioned any dosage and therapeutic scheme can use.For example, a suitable scheme is, every month to serve as intravenous administration at interval, dosage range is the antibody of 0.1-1.0mg/kg or the antibody of preferred 0.5-1.0mg/kg.For subcutaneous administration, used dosage usually in the scope of 0.01-0.6mg/kg or 0.01-0.35mg/kg, preferred 0.05-0.25mg/kg.For once in a week or administration biweekly whenever, dosage is preferably in the scope of 0.015-0.2mg/kg or 0.05-0.15mg/kg.For weekly administration, dosage is preferably 0.05-0.07mg/kg, for example, and 0.06mg/kg.For administration biweekly whenever, dosage is preferably 0.1-0.15mg/kg.For every month single administration, dosage was preferably 0.1-0.3mg/kg or 2mg/kg.

Here and the application's other places, the quality that the dosage of representing with mg/kg can be by multiply by typical patient (for example, 70 or 75kg) converts to absolute mass dosage, is rounding to integer usually.Represent that according to absolute mass antibody is pressed the dosage of 1-40mg usually, with weekly extremely mensal frequency administration.Preferable range is 5-25mg or 2.5-15mg, and frequency is once in a week to every month once.To administration biweekly whenever, dosage usually is 1-12mg or 2.5-10mg for weekly.For weekly administration, dosage usually is 2.5-5mg or 4-5 mg.For administration biweekly whenever, dosage can be 7-10mg.Antibody quality with the unit dose packaging administration is rounding to integer usually, as 1,5,10,20,30,40,50,75 or 100mg.

The dosage of administration and frequency can be preventative or curative the variations according to treatment.In prophylactic use, do not use and contain this antibody or with the compositions of its mixture, to strengthen patient's resistance to also being in patient in the morbid state.Such amount is defined as " effectively preventive dose ".In this application, accurate amount still depends on patient's health and general immunity situation, but usually in the scope of every dose of 0.1-25mg, particularly every dose of 0.5-2.5mg.Low relatively dosage is with the not frequent long-time administration in interval relatively.Some patients continued to receive treatment in its remaining years.

In therapeutic is used, sometimes need be with the relative high dose (for example, every dose of about 10-250mg antibody, the dosage of 5-25mg more commonly used) at short relatively interval, until the progress that alleviates or stop disease, preferably show the partially or completely improvement of disease symptoms until the patient.Afterwards, can use preventative scheme to the patient.

Medicament of the present invention can be chosen wantonly with other and become in the treatment of diseases to small part efficacious agents combination medicine-feeding at amyloidogenic.Under amyloid deposition appears at CAA situation in the cerebrovascular system, medicament of the present invention can also with increase medicament of the present invention other medicament administering drug combinations by blood brain barrier.

The dosage range of immunogenic nucleic acid of encoding is about 10ng to 1g, 100ng-100mg, 1 μ g-10mg or 30-300 μ g DNA/ patient.The dosage of venereal infection poisonous carrier is from every dose of 10-100 or more virion and different.

The medicament of induce immune response can pass through in parenteral, part, intravenous, oral, subcutaneous, intra-arterial, intracranial, the sheath, intraperitoneal, intranasal or the administration of intramuscular mode, is used for preventative and/or therapeutic treatment.Although other approach is effective equally, the most typical route of administration of immunogenic agents is subcutaneous.Secondly modal approach is intramuscular injection.Such injection is the most common to be carried out in arm or leg muscle.In certain methods, medicament is injected directly in the cumulative particular organization of deposit, for example, and intracranial injection.Intramuscular injection or venoclysis are that antibody administration institute is preferred.In certain methods, specific therapeutic antibodies is injected directly into intracranial.In certain methods, antibody is as slow releasing composition or device, as Medipad ^TMDevice is used.

As indicated above, inducing separately can combination medicine-feeding to the medicament of the immunogenic response of A β.Medicament can be combined into unitary agent or kit, with simultaneously, use in succession or separately.Medicament can take independent bottle in preparation or examination kit, perhaps can be merged into single bottle.These medicaments of the present invention can choose wantonly with other in the treatment of CAA to small part efficacious agents combination medicine-feeding.Portugal's amine polysaccharide immitation (glycosaminoglycan-mimetic) CEREBRILL (Neurochem) is in the clinical trial of treatment CAA at present.Should advise that most of CAA patients avoid " make blood thin " or disturb the medicament of blood coagulation.Influencing the strongest medicine of blood coagulation (therefore the most dangerous to CAA patient) is warfarin (also claiming its trade name " Coumadin ").Other has aspirin, ticlopidine (" Ticlid "), clopidogrel (" Plavix ") and most of anti-inflammatory drug such as ibuprofen to the more weak medicine of blood influence.And the patient recovers the common careful monitoring blood pressure in back at hemorrhagic apoplexy, and it was remained in normal range.Epilepsy or the recurrent nervous symptoms that is thought of as epilepsy are used antuepileptic and are handled, although Depakote (sodium valproate) is because its antiplatelet effects and should avoiding.Sometimes it is still unclear with the benefit of attempting to prevent epilepsy, even now and doing to give antuepileptic to the patient of a large amount of cerebral lobar hemorrhages.Eliminate cerebral hemorrhage and may need operation.CAA is rare in cerebral vasculitis or vessel wall inflammation.In these cases, has help with steroid or immune system inhibitor.

Immunogenic agents of the present invention is as peptide, sometimes with the adjuvant combination medicine-feeding.Many kinds of adjuvants may be used to and the peptide combination, to cause immunne response.Preferred adjuvant strengthens immunogenic intrinsic replying, and does not cause the immunogen conformation change of the character form that influence is replied.Preferred adjuvants comprises aluminium hydroxide and aluminum phosphate, 3 De-O-acidylate monophosphoryl lipid A (MPL ^TM) (see GB 2220211 (Montana now is the part of Corixa for RIBI ImmunoChem Research Inc., Hamilton).Stimulon ^TMQS-21 is that the Triterpene Glycosides or the saponin that separate soapbark (the Quillaja Saponaria Molina tree) bark of finding from South America (are seen Kensil etc., (Powell and Newman write Vaccine Design:The Subunit and Adjuvant Approach (vaccine design: subunit and adjuvant approach), Plenum Press, NY, 1995); U.S. Patent No. 5,057,540, Aquila BioPharmaceuticals, Framingham, MA.Other adjuvant has oil in water emulsion (as Squalene or Oleum Arachidis hypogaeae semen), its optional and immunostimulant combination, as monophosphoryl lipid A (seeing Stoute etc., N.Engl.J.Med.336,86-91 (1997)), the general mycobacteria of flowing Buddhist nun's gram (pluronic) polymer and having killed.Another adjuvant is CpG (WO98/40100).Perhaps, A β can with the adjuvant coupling.Yet to regard to the immunogenic immunne response character, such coupling should not change immunogenic conformation substantially with regard to influence.Adjuvant can be used as the composition administration with the therapeutic combination of activating agent, perhaps can be before the therapeutic agent administration, simultaneously and individually dosed afterwards.

The preferred adjuvants kind is aluminum salt (vitriol), as aluminium hydroxide, aluminum phosphate, aluminum sulfate.Such adjuvant can with or not with other specific immunostimulant such as MPL or 3-DMP, QS-21, amino acid polymer or monomer such as polyglutamic acid or polylysine use together.Another kind of adjuvant is the oil in water emulsion preparation.Such adjuvant can with or not with other specific immune stimulant such as muramyl peptide (for example; N-acetyl muramyl-L-Threonyl-D-isoglutamine (thr-MDP); N-acetyl group-fall muramyl-L-alanyl-D-isoglutamine (nor-MDP); N-acetyl muramyl-L-alanyl-D-isoglutamine acyl group-L-alanine-2-(1 '; 2 '-two palmityls-sn-glycerol-3-hydroxyl phosphorus acyloxy)-and ethamine (MTP-PE), N-acetyl glucosamine base-N-acetyl muramyl-L-Al-D-isoglutamic acid-L-alanine-two Petiolus Trachycarpi oxygen base propionic acid amide. (DTP-DPP) theramide ^TM) or other bacteria cell wall composition use together.Oil in water emulsion comprises (a) MF59 (WO 90/14837), contain 5% Squalene, 0.5% Tween 80 and 0.5%Span 85 (choose wantonly and contain not commensurability MTP-PE), with microfluidization device such as 110Y type microfluidization device (Microfluidics, Newton MA) makes submicron particle, (b) SAF, contain 10% Squalene, 0.4% Tween 80,5% general stream Buddhist nun and restrain block polymer L121 and thr-MDP, all miniflow changes into submicron Emulsion or vortex to producing the bigger Emulsion of granularity, and (c) Ribi ^TMAdjuvant system (RAS), (Ribi ImmunoChem, Hamilton, MT) contain 2% Squalene, 0.2% Tween 80 and from one or more bacteria cell wall compositions of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferred MPL+CWS (Detox ^TM).

Another kind of preferred adjuvants is the saponins adjuvant, as Stimulon ^TM(Framingham is MA) or by the granule of its generation, as ISCOMs (immunostimulating complex) and ISCOMATRIX for QS-21, Aquila.Other adjuvant comprises RC-529, the incomplete Freund's adjuvant (IFA) of GM-CSF and pharmaceutically acceptable level (selling with trade name Montanide).Other adjuvant comprises cytokine, as interleukin (for example, IL-1, IL-2, IL-4, IL-6, IL-12, IL-13 and IL-15), M-CSF (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor (TNF).Another kind of adjuvant is a glycolipid analogs, comprises N-glycosyl amide, N-glycosyl urea and N-glycosyl carbamate, on its each comfortable saccharide residue by aminoacid replacement, as immunomodulator or adjuvant (seeing U.S. Patent No. 4,855,283).Heat shock protein, for example HSP70 and HSP90 also can be used as adjuvant.

Adjuvant can be with immunogen as single compositions administration, perhaps can be before the immunogen administration, simultaneously or administration afterwards.Immunogen and adjuvant can be packed and be supplied in the same bottle, perhaps can be packaged in the bottle separately, mix before the Yu Linyong.The label that immunogen and adjuvant conventional packing have the explanation predetermined treatment to use.If immunogen and adjuvant are packed respectively, packing generally includes faces with preceding blended description.The stability, route of administration, administration time table, adjuvant that the selection of adjuvant and/or carrier depends on the immunogenic formulation that contains adjuvant is to by the effectiveness of inoculation species, and, for the people, pharmaceutically useful adjuvant is maybe can be ratified to be used for adjuvant to people's administration by related management department approval.For example, complete Freund's adjuvant is not suitable for the administration to the people.Vitriol, MPL and QS-21 are preferred.Choose wantonly, can use two or more different adjuvants simultaneously.Preferred compositions comprises vitriol and MPL, vitriol and QS-21, and MPL and QS-21, MPL or RC-529 and GM-CSF, and vitriol, QS-21 and MPL are together.Also can use incomplete Freund's adjuvant (Chang etc., Advanced Drug DeliveryReviews 32 (advanced drug delivery summary 32), 173-186 (1998)), any one and all compositions among optional and vitriol, QS-21 and the MPL is combined.

Medicament of the present invention promptly, comprises active treatment medicament and multiple other pharmaceutically acceptable composition usually as the pharmaceutical composition administration.See Remington ' s PharmaceuticalScience (Lei Shi pharmaceutical science) (the 15th edition, Mack Publishing Company, Easton, Pennsylvania, 1980).Preferred form depends on the preassigned pattern that administration and treatment are used.According to required preparation, compositions can also comprise pharmaceutically acceptable non-toxic carrier or diluent, and it is defined as the medium that is commonly used to prepare to the pharmaceutical composition of animal or human's administration.Select diluent not influence the biological activity of compositions.The example of such diluent has distilled water, physiology phosphate buffered saline (PBS), Lin Ge (Ringer ' s) solution, dextrose solution and Hank solution.In addition, pharmaceutical composition or preparation can also comprise the stabilizing agent of other carrier, adjuvant or nontoxic, no therapeutic, non-immunogenicity etc.

Pharmaceutical composition can also comprise big metabolism macromole slowly, as protein, polysaccharide such as chitosan, polylactic acid, polyglycolic acid and copolymer (agarose functionalized (TM), agarose as latex, cellulose etc.), polymeric aminoacid, amino acid copolymer, and Liposomal assembly (as oil droplet or liposome).In addition, these carriers can play the effect of immunostimulant (that is adjuvant).

For parenteral, medicament of the present invention can be with the material solution in acceptable diluent and the pharmaceutical carrier or injectable dosed administration of suspension on the physiology, and described pharmaceutical carrier can be a sterile liquid, Ru Shui, oil, saline, glycerol or ethanol.In addition, complementary material as wetting agent or emulsifying agent, surfactant, pH buffer substance etc., may reside in the compositions.Other composition of pharmaceutical composition is those of oil, animal, plant or synthetic source, for example Oleum Arachidis hypogaeae semen, soybean oil and mineral oil.Generally speaking, glycol such as propylene glycol or Polyethylene Glycol are preferred fluid vehicles, especially for injection.Antibody can be with the storage injection (depot injection) prepared according to the mode that allows active component to continue to discharge or the form administration of implantation preparation.Exemplary composition comprises the 5mg/mL monoclonal antibody, prepares in the water-containing buffering liquid of being formed, be adjusted to HCl pH 6.0 by 50mM L-histidine and 150mM NaCl.The compositions that is used for parenteral is aseptic basically usually, and first-class substantially, and produce according to the GMP condition of FDA or similar department.For example, the compositions that contains biological agent is sterilized with sterilization by filtration usually.Compositions can be mixed with and be used for single dose administration.

Usually, preparation of compositions is become the injectable agent of liquid solution or suspension; Also can be prepared into the solid form of in liquid medium, making solution or suspension before being suitable for injecting.As discussed above, for strengthening adjuvant effect, can also with preparation emulsifying be encapsulated in liposome or microgranule such as polylactide, poly-Acetic acid, hydroxy-, bimol. cyclic ester or copolymer in (see Langer, Science 249,1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28 (advanced drug delivery summary 28), 97-119 (1997)).Medicament of the present invention can be to store the form administration of injection or implantation preparation, and they can be by allowing the mode that active component continues or pulsed discharges to prepare.

That other preparation that is suitable for other administering mode comprises is oral, intranasal and lung preparation, suppository, and transdermal is used.

For suppository, binding agent and carrier comprise, for example, and poly alkylene glycol or triglyceride; Such suppository can be made with the mixture of the active component that contains 0.5%-10%, preferred 1%-2% scope.Oral formulations comprises excipient, as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose and magnesium carbonate.These compositionss are taked the form of solution, suspension, tablet, pill, capsule, slow releasing preparation or powder, and contain the active component of 10%-95%, preferred 25%-70%.

Topical application can cause transdermal or Intradermal to be sent.Medicament and cholera toxin or its detoxification derivant or subunit or other similar bacteriotoxin co-administered can promote topical (see Glenn etc., Nature 391,851 (1998)).Co-administered can be by finishing as mixture or as the link molecule that obtains through chemical crosslinking or as being expressed as fusion rotein with described composition.

Perhaps, transdermal delivery can paste medicament or finish (Paul etc., Eur.J.Immunol.25,3521-24 (1995) with conveying body (transferosomes) with skin; Cevc etc., Biochem.Biophys.Acta 1368,201-15 (1998)).

VI. kit

The present invention further provides the treatment product.Product comprises vial and description.Vial contains preparation, and said preparation comprises that about 10mg is to about 250mg humanization anti-amyloid beta antibodies, about 4% mannitol or about 150mM NaCl, about 5mM to about 10mM histidine and about 10mM methionine.Description comprises by MRI monitoring patient's brain little hemorrhage, perhaps monitors the removal of patient's blood vessel amyloid by PET scanning.

Embodiment

Embodiment 1

Material and method

Research design.In two researchs, investigate chronic passive immunity to the PDAPP mice in the influence of established VA β.The A research design is become to compare single agent effectiveness of N-terminal antibody (3D6, identification A β 1-5) and mesozone antibody (266, identification A β 16-23).B research is the dose response studies of 3D6.In two researchs, the female heterozygosis PDAPP mice at 12 monthly ages is divided into 40 one group; Each group is mated close on age and transgenic parent as far as possible.In separately assessing, at t ₀The time put to death one group of 40 animal, the blood vessel amyloid level when measuring for 12 monthly ages.Such as table 1 summary, the processed group mice is by peritoneal injection mouse monoclonal antibody 3D6 γ 2a (with 3 kinds of dosage levels), 266 γ 1 or TY11-15 (as negative control).All processed animals all are accepted as 250% the initial load dosage of planning weekly dosage.The dosage of every animal calculates according to the historical average weight 50g of this age bracket PDAPP mice.Animal is handled weekly once, altogether about 6 months (26 week).After survival period (in-life phase) stops, estimate VA β and little hemorrhage existence and degree.All working all carries out according to Elan IACUC guideline.

The preparation of antibody.Before existing description of preparation method of antibody 3D6 (identification A β 1-5), 266 (identification A β 16-23) and 12A11 (identification A β 3-7) (seen K.Johnson-Wood etc., Proc Natl Acad Sci USA 94,1550-5 (1997), P.Seubert etc., Nature 359,325-7 (1992), F.Bard etc., Proc Natl Acad Sci USA 100,2023-8 (2003)).TY11/15 (IgG _2aIsotype) plays irrelevant control antibodies.The human lymphocyte antigen that its identification is unknown, but nonrecognition mouse lymphocyte.Antibody 3D6 and 12A11 are as discussed previously with NHS-biotin labeling (see P.Seubert etc., Nature 359,325-7 (1992)).

The preparation cerebral tissue is used for histochemistry.Animal is used the isoflurane deep anaesthesia, perfusion saline in the ventricle.With a hemisphere of each brain in 4% paraformaldehyde in 4 ℃ of submerged fixed 48 hours, on vibratome, cut into slices with 40 μ m coronalplanes.Before the immunostaining, (30% glycerol/30% ethylene glycol is at the 40mM of pH 7.4 Na section to be stored in-20 ℃ anti-icing fluid ₂HPO ₄In) in.Each brain is selected 4-6 section, crosses over kiss side Hippocampus level with the interval of 240 μ m, analyzes.Impaired brain and the section of volume cortex will not be analyzed when taking out from skull.Final numbering is presented among the result.Section is dyeed by the researcher of not knowing disposition and is analyzed.

VA β and little hemorrhage histochemistry program of labelling altogether: A β deposit is spent the night in 4 ℃ of labellings in the PBS of 1% horse serum solution with biotinylated antibody 3D6 (3.0 μ g/ml) or 12A11 (3.0 μ g/ml).Buoyant section is reacted with Avidin-biotinylation horseradish peroxidase complex then, and with 3, the 3-diaminobenzidine develops.Then section is fixed on the microscope slide, is total to dyeing (co-stained) (seeing M.M.Racke etc., the same) with the Perls ferrum reaction that improves, improvement is to hatch at 37 ℃, to strengthen the hemosiderin product.The existence of hemosiderin shows that there is little bleeding episode in the past.

VA β analyzes: by animal classification is become to reflect one of 2 classes that VA β measures:: " not having to a little VA β " (in any single section≤3 amyloid positive vessels/animals) or " moderate VA β " (in any single section＞and 3 amyloid positive vessels/animals), the 3D6 immunoreation blood vessel in each animal is estimated.This sorting technique is by counting the proteic blood vessel of all starch-containing samples in the tissue of A and B research, and identifies balance sensitivity and specific best cutoff is set up with the ROC curve.If blood vessel contains the amyloid of any amount, just to its counting, like this, blood vessel of partly being removed and the blood vessel that is not eliminated are just all counted.Adopt Fisher definitely to check (FET) to carry out paired comparison, to identify the significant difference of VA β.In each research, adjust multiplex paring relatively with Hochberg method (see Y.Hochberg, Biometrika 75,800-802 (1988)).

Littlely go out haemanalysis: each animal painted existence of hemosiderin, amount, position and intensity in whole section are carried out the scoring of 0-3 level." 0 " divides expression dyeing considerably less or dye-free, " 1 " represents that each animal has speckle or weak dyeing in a small amount in the minority section, " 2 " are meant the continuous accumulations that has staining power higher in a plurality of sections, " 3 " are reflected in and have observed deep dyed color in the great majority sections, generally include most periphery and are attacked blood vessel.The design of these grades is limited to the scope of the hemosiderin positive staining of this clinical preceding zooscopy of reflection, therefore can not represent or convert to the grade of clinical hemorrhagic disease.Adopt the definite check of Fisher to carry out paired comparison, with the difference between the check processed group.Also observe to the morphology outward appearance of immune labeled amyloid and with the spatial relationship of hemosiderin.In each research, adjust multiplex paring relatively with Hochberg method (seeing Y.Hochberg, the same).

The result

Blood vessel A β.The thioflavin S dyeing of fine and close amyloid (Fig. 1 a) and the antibody 3D6 (Fig. 1 b) of densification among identification PDAPP mice and the people AD (Fig. 1 c) and diffusivity amyloid disclose, VA β is remarkable in pia arachnoid of untreated 18 monthly age PDAPP mices and the shallow table essence.VA β mainly is confined to meninges, and abuts against (Fig. 1 d) below the shallow top layer of brain.In cortex, especially remarkable especially in the center line blood vessel of sagittal sinus, thioflavin S (Fig. 1) and antibody 3D6 (Fig. 1 b) show to have similar VA β to distribute in mice and people's tissue (Fig. 1 c).

In the single dose comparative study (A research) of N-terminal and mesozone antibody, with 266 or TY11-15 relatively, the 3D6 of 3mg/kg removes fully or has stoped VA β (Fig. 2 a-e); These differences have significance (two comparisons, p value are all＜0.0001) statistically.3D6 handles and has also reduced by 98% essence amyloid plaques loading (p＜0.0001), and 266 not generation effects.Before beginning to handle, VA β is present in medium level in 23% the 12 monthly age mices.Adopt the Hochberg method of multiple comparisons, FET p value＜0.025 has significance statistically.

In the dose response studies of 3D6 (B research, Fig. 3 a-d), compare with the TY11-15 control treatment, 3D6 handles and significantly removes on the 3D6 of 3.0mg/kg dosage level once more or stoped VA β (p＜0.001).Compared with the control, middle dosage level (0.3mg/kg) is also removed or has been stoped VA β (p=0.016).0.1mg/kg zero difference ((p=0.8037) between dosage group and TY11-15 matched group.Adopt the Hochberg method of multiple comparisons, FET p value＜0.025 has significance statistically.Although blood vessel is counted there was no significant difference, this group on the microscope level, observed the part of the amyloid of blood vessel remove (Fig. 4 a, b).Though shown in Fig. 4 a, complete A β forms and surrounds the piece and the band of being attacked the pia arachnoid blood vessel, and when part was removed, VA β had the outward appearance that is corroded (Fig. 4 b) of fritter.This form does not see untreated mice.

The hemosiderin evaluation.For distinguishing the nuance between processed group, formulated the hemosiderin rating scale, reflect the scope of the stain density of finding in this research.Indicate little hemorrhage hemosiderin dyeing to be limited to the structure boundary of vascular system and not extend to essence on every side.The minority essence blood vessel that local hemosiderin deposit sees the sagittal sinus blood vessel of cortex pia arachnoid (Fig. 5 a-f) and the Hippocampus-blood vessel at thalamus interface, inboard cortex, meets at right angles with the pia arachnoid blood vessel and be connected.Hemosiderin concentrates in these regional macrophages usually.The normal form with VA β of hemosiderin kitchen range changes together: with the sedimentary characteristic of VA β unique be with and small pieces (scales) (for example, Fig. 4 is difference a), and amyloid has uncommon speckle shape and degraded sample outward appearance (for example, Fig. 4 b) or do not exist fully.These features are especially remarkable in the blood vessel of pia arachnoid (Fig. 5) and sagittal sinus, and it often demonstrates the VA β form that has developed in TY11-15 matched group and untreated mice (Fig. 2 and 3).

In all processed group, hemosiderin dyeing generally lacks or is slight, and the most of animal scorings between group are 0 or 1 (Fig. 6).The scoring of the 3D63mg/kg processed group of two researchs is obviously higher, shows that these comparisons are according to the hemosiderin scoring that more likely has greater than 0.In A research, the TY11-15 group is similar to 266 groups hemosiderin distribution of grading, shows that 266 antibody treatment can not improve the hemosiderin scoring.B studies show that little hemorrhage incidence rate has reduced by administration.Do not find significant difference between TY11-15 matched group and 0.1mg/kg 3D6 group and the 0.3mg/kg 3D6 group, show that low dosage and middle dosage can not make the hemosiderin evaluation increase on the baseline values.3.0mg/kg organize different with these groups, its also with the contrast there were significant differences.Compare with A research, the higher hemosiderin scoring of 3.0mg/kg group may be owing to antibody exposure level difference in time, and the baseline values of group has slight increase because of the scoring of matched group improves a little.

VA β removes and little hemorrhage relation.Study remarkable removing or the prevention of all in 0.3mg/kg and 3.0mg/kg group, having observed the blood vessel amyloid for two.The hemosiderin scoring of most of animals in these groups is 0 or 1, shows that most of brains that VA β reduces all have seldom or do not have little hemorrhage sign.In all processed group, found to have very odd VA β and several examples (for example, Fig. 4 b) of the negative blood vessel of the hemosiderin of the sample outward appearance that is corroded; These may be that amyloid is not having little hemorrhage blood vessel that is eliminated down.When observing hemosiderin dyeing in the animal (Fig. 5 d) that 0.1mg/kg 3D6 handles, it is accompanied by speckle shape amyloid usually in circumvascular distribution, may show the removing well afoot.These circumvascular amyloid specklees appear at the position of the relevant hemosiderin labelling of the endovascular blood vessel of cortex meninges, essence and sagittal sinus.0.3mg/kg 3D6 handles the hemosiderin dyeing of animal and also is accompanied by speckle shape amyloid at circumvascular distribution (Fig. 5 e).The form of amyloid is handled the similar of animal to 0.1mg/kg 3D6, but amyloid is not abundanter, and some hemosiderin positive vessels have been eliminated amyloid.Comprise observed on the painted position of the relevant hemosiderin of blood vessel of cortex meninges and essence amyloid fully and part remove.On the contrary, the hemosiderin positive vessels of 3mg/kg 3D6 processing animal does not usually have amyloid (Fig. 5 f) fully.This feature is never observed in untreated mice, may illustrate to appear at the residual hemosiderin " footprint " that exists in the subgroup blood vessel that VA β removed fully.These regional another feature are to exist born of the same parents to bite the cell (Fig. 5 f) of hemosiderin.These macrophages do not have immunoreation to A β, therefore seemingly are different from the microglia of the relevant amyloid of removal speckle and the colony of macrophage.

Discuss

CAA has been accredited as cognitive impaired independent risks and assumptions, itself and important pathological such as hemorrhage relevant with ischemic injury (see S.M.Greenberg etc., Stroke 35,2616-9 (2004)).In model case, progressivity CAA has caused the destruction of the smooth muscle cell in meninges and the essence vascular system, infer and caused the disease damage (tonal impairment) and the infringement of scavenging system on every side of perfusion and blood vessel (to see R.Christie etc., Am J Pathol 158,1065-71 (2001), S.D.Preston etc., Neuropathol Appl Neurobiol 29,106-17 (2003)).

Here, we have showed for the first time with in the peripherally administered permanent immunity treatment example of antibody, the evidence that VA β is almost completely removed or stops by N-terminal specificity A β antibody (3D6).Although understanding mechanism is not that enforcement is essential to the invention, but this effect may depend on securely the ability in conjunction with sedimentary amyloid because in vivo in conjunction with a little less than the ability of sedimentary A β many mesozone A β antibody (266) does not demonstrate the evidence of removing or stoping VA β.Though there is more and more evidences to show, the formation of blood vessel amyloid may differently with the essence speckle (be seen M.C with forming, Herzig etc., Nat Neurosci 7,954-60 (2004)), but antibody 3D6 has the ability to remove these two kinds of forms, therefore has the activity that broad-spectrum reduces amyloid.

Previous effect and the cerebral microvascular systematic research of investigating A β immunization therapy in the APP transgenic mice reported that little hemorrhage incidence rate increases.Yet, description is not also arranged for VA β and little clear and definite cause effect relation between hemorrhage.In this report, we show that most of sedimentary blood vessel amyloid is eliminated, and do not induce little hemorrhagely, and relevant with the removal of VA β by the little hemorrhage incidence rate of proof, have improved early stage observation.In addition, little hemorrhage finite region is concentrated and is confined to the structure boundary that vascular system not relates to essence.This partially or completely removal with VA β is relevant.Importantly, can still effectively remove in the scope of essence amyloid plaques, significantly alleviate little hemorrhage by antibody dosage is adjusted to.

Racke and colleague (seeing M.M.Racke etc., the same) report, the PDAPP mice is seldom taken place little hemorrhage with after obviously more heavy dose of 3D6 handled for 6 weeks.Little hemorrhage degree that it should be noted that report is also greater than observed any incident in the current research, and this is consistent about positively related discovery between antibody dosage and the little hemorrhage scoring with us.Our observed result can not or induce little hemorrhage discovery consistent with them in conjunction with sedimentary amyloid about 266, and the extension of these discoveries has shown that 266 can not remove VA β.

In the very serious APP transgenic mice of VA β pathology (APP23 mice), the spontaneous generation of cerebral hemorrhage, and, similar to human patients, this may be that smooth muscle cell is disorderly and lose and result's (seeing R.Christie etc., the same) of other destructive consequence that A β is xicity related.The APP23 mice has aggravated hemorrhage generation and the degree (see M.Pfeifer etc., Science 298,1379 (2002)) of baseline at first with N-terminal district A β antibody passive immunity.Yet ultrastructural studies is not subsequently found architectural difference (seeing G.J.Burbach etc., the same) in the vascular system of processed and non-immune contrast.Conclusion is, no matter the severity of baseline VA β pathology, immunization therapy does not cause or increases the weight of obvious damage to blood vessel wall.What this research and previous report different were to investigate is seldom spontaneous little hemorrhage model, and wherein A β and hemosiderin by labelling altogether.We have write down A β and have removed and little hemorrhage common location, little hemorrhage the occurring over just in the subgroup blood vessel that is eliminated amyloid of concurrent current situation portion.Because our quantitative approach can not be distinguished VA β and complete VA β that part is removed, therefore absolute removing degree may be underestimated.

Relation between the removing of essence amyloid and blood vessel amyloid is not also understood fully.Yet recent report shows, may have common adjusting relation before two kinds of pathology, this can in the content of removing speckle, further illustrate (see D.M.Wilcock etc., the same; M.C.Herzig etc., the same; J.A.Nicoll etc., J Neuropathol Exp Neurol 65,1040-8 (2006)).For example, in fact the mice of the sudden change APP mice of serious VA β being raised into serious essence speckle loading has reduced VA β, and the prompting speckle can provide template for A β, otherwise it will be deposited on the vascular system and (sees M.C.Herzig etc., the same).Conversely, Wilcock and colleague (seeing D.M.Wilcock etc., the same) show that in the passive immunity example, VA β increases in the removal process of essence speckle, prompting in the immunization therapy process, may take place A β from substantial displacement to lumen of vessels.

In this research, we have proved that VA β can almost completely be removed or stop behind passive immunity, and the little hemorrhage incidence rate increase of being followed can reduce by antibody administration.The APP transgene mice model of AD patient and AD shows that all the increase of little hemorrhage incidence rate is relevant with the progress of VA β.Described hereinly littlely hemorrhagely may increase (seeing D.M.Wilcock etc., the same) in the process of removing the essence speckle by the described VA β of Wilcock and make an explanation, it expect in this model is temporary transient, because during to this research end, VA β is finally removed.Perhaps, in the research of Wilcock, used different mouse models and antibody, so the variation of VA β can reflect the fundamental mechanism difference about different antibodies epi-position and animal model.In any case, suppose to remove already present VA β and will further little hemorrhage the have preventive effect relevant with progressivity VA β then in fact be reduced little hemorrhage final accumulation and take place seemingly possible after the processing of 3D6 with the prevention further precipitation.In other words, handle relevant VA β and VA β subsidiary (VA β-contingent) little hemorrhage may be transient phenomenon, this phenomenon will be no longer lasting after VA β is by final the removal.Generally speaking, the discovery that is obtained by preclinical models shows that the mechanism relevant with removing with the formation of VA β also needs further research.Importantly, recent research shows, in fact the integrity that has improved blood brain barrier with total length A β peptide immunity TG 2576 mices (promptly, reduced the permeability of azovan blue), point out these multiple factors in fact may have positive impact (seeing D.L.Dickstein etc., the same) to vascular system.It should be noted that the immunity of complete A β peptide has produced the antibody that is primarily aimed at the N-terminal similar to the epi-position of 3D6.

About 80% AD patient is subjected to the invasion and attack of at least slight CAA, and that clinical harmful consequence has is hemorrhage, white matter degeneration, ischemia and inflammation (seeing S.M.Greenberg etc., the same).Our discovering provides the A β immunization therapy can potential reverse or stop the evidence of the important blood vessels pathology progress that does not also have Therapeutic Method at present, and further expanded the potential treatment interests of anti-A β immunization therapy.

Embodiment 2

Material and method

Investigated the influence of the inductive structural change of amyloid, and passive immunity is to the SMC of PDAPP mice blood vessel and the influence of ECM to the smooth muscle cell (SMC) and the extracellular matrix (ECM) of PDAPP mice blood vessel.

Once in a week with 1 or the 3D6 antibody of 3mg/Kg mice is carried out immunity, by a definite date 3 or 9 months.To VA β deposit significantly the meninges blood vessel of the sagittal sinus of (about 70% blood vessel is attacked) carry out vascular components (to SMC be alpha Actinin, is that the quantitative immunohistochemistry of high-resolution (IHC) of collagen-IV) is analyzed to ECM.Monitor little bleeding episode by hemosiderin detection or ferritin immunohistochemistry.

The result

In this research, we have proved that the variation of blood vessel wall is always relevant with VA β, and it comprises degeneration (thickness minimizing) and the hypertrophy/hypertrophy (thickness increase) of SMC and ECM.The result of these two kinds of opposition is common in same blood vessel, but is not present in the wild type animal or does not have in the PDAPP blood vessel of amyloid.Be untreated in the PDAPP mice, the extreme of SM thickens with attenuation and causes significantly variable blood vessel phenotype.

Passive immunotherapy has recovered the thickness mode of vascular SMC and ECM in the mode of dosage and time dependence, and has reduced the transmutability of phenotype, and the 3D6 of high dose has reached control level (wild type) (p＞0.05) in the time of 9 months.Though little hemorrhage incidence rate increased in the group at 3 months, it has been reduced to control level (p＞0.05) after 9 months processing.Our results suggest, passive immunotherapy can make the meninges blood vessel recover from the inductive structural change of amyloid.In addition, handle relevant little hemorrhage increase transient event seemingly, it disappears in the process that VA β removes.The mechanism of repairing may be triggered by the removal of VA β, finally causes the recovery of dysfunction of blood vessel.

Although foregoing invention is described in detail for the clear purpose of understanding, it is evident that, can implement some modification within the scope of the appended claims.All publications that this paper quoted all are incorporated herein by reference with identical degree for all purposes in full with patent documentation and appearance text in the accompanying drawings, just as what pointed out individually separately.

Claims (106)

1. the method for treatment CAA comprises

To suffering from or suspect that the patient who suffers from CAA uses the medicament of effective scheme, wherein said medicament is that the N-terminal to A β has specific antibody or inducing such antibody after patient's administration, thus and treatment patient.

2. the process of claim 1 wherein that medicament is an antibody.

3. the method for claim 2, wherein medicament is the intra-residue antibody in 1-5 position that is combined in A β.

4. the method for claim 2, wherein antibody is humanized antibody, people's antibody or chimeric antibody.

5. the method for claim 4, wherein antibody is humanization 3D6 or humanization 12A11.

6. the method for claim 5, wherein the 3D6 humanized antibody is a Ba Biniu pearl monoclonal antibody (bapineuzumab).

7. the process of claim 1 wherein that medicament is the fragment of A β.

8. the method for claim 7, wherein fragment starts from 1 residue of A β, and terminates in one of 5-10 position residue of A β.

9. the method for claim 7, wherein fragment is A β 1-7.

10. the method for claim 7, wherein the fragment of A β is with pharmaceutically useful adjuvant administration.

11. the method for claim 7, wherein the fragment of A β induces the carrier at this segmental antibody to be connected with helping this fragment.

12. the method for claim 11, wherein carrier is connected with segmental C-terminal.

13. the method for claim 1 comprises also and determines that the patient suffers from CAA that wherein determining step occurs in before the dosing step.

14. the method for claim 13, wherein determining step determines that the patient suffers from the clinical symptoms of CAA.

15. the process of claim 1 wherein and not have the characteristic speckle of Alzheimer's disease in patient's brain.

16. the method for claim 14, wherein the patient does not have the symptom of Alzheimer's disease.

17. the process of claim 1 wherein that the patient had heart attack or apoplexy.

18. the process of claim 1 wherein that the dosage of antibody is between about 0.01 to about 5mg/kg.

19. the process of claim 1 wherein that the dosage of antibody is between about 0.1 to about 5mg/kg.

20. the method for claim 18, wherein dosage is about 0.5mg/kg.

21. the method for claim 18, wherein dosage is about 1.5mg/kg.

22. the method for claim 18, wherein dosage is between about 0.5 to about 3mg/kg.

23. the method for claim 18, wherein dosage is between about 0.5 to about 1.5mg/kg.

24. the method for claim 18 is wherein repeatedly used described dosage.

25. the method for claim 18 is wherein weekly to the described dosage of per season applied once.

26. the method for claim 18, wherein per 13 weeks are once used described dosage.

27. the process of claim 1 wherein that antibody is through intravenous or subcutaneous administration.

28. the method for claim 1 also comprises the variation of monitoring at the S or S of the CAA response of dosing step.

29. the method for claim 1 also comprises second medicament of using effective treatment CAA.

30. realize the method for prevention CAA, comprise

Use the medicament of effective scheme to the patient of easy trouble CAA, wherein said medicament is that the N-terminal to A β has specific antibody or inducing such antibody after patient's administration, thereby and realizes patient's prevention.

Have specific antibody or after patient's administration, inducing such antibody 31. medicament application in the Alzheimer's disease in treatment or prevention, wherein said medicament are the N-terminal to A β.

32. reduce the method for patient vessel's amyloid, comprise

The N-terminal of using A β according to the therapeutic scheme with reducing the little hemorrhage incidence rate of brain relevant with effective removal blood vessel amyloid has specific antibody.

33. the method for claim 32 also comprises

Brain by MRI monitoring patient is little hemorrhage.

34. the method for claim 32 also comprises

Monitor the removal of patient's blood vessel amyloid by PET scanning.

35. the method for claim 32, wherein therapeutic scheme is a long-term treatment regimen.

36. the method for claim 32, wherein therapeutic scheme comprises weekly to the antibody dosage of per season applied once between 0.01 to 5mg/kg weight in patients.

37. the method for claim 36, wherein the dosage of antibody is 0.1-5mg/kg.

38. the method for claim 36, wherein dosage is about 0.5mg/kg.

39. the method for claim 36, wherein dosage is about 1.5mg/kg.

40. the method for claim 36, wherein dosage is between about 0.5 to about 3mg/kg.

41. the method for claim 36, wherein dosage is between about 0.5 to about 1.5mg/kg.

42. the method for claim 36, wherein per 13 weeks are once used described dosage.

43. the method for claim 32, wherein antibody is through intravenous or subcutaneous administration.

44. the method for claim 32, wherein antibodies is in the residue of the 1-5 position of A β.

45. the method for claim 32, wherein antibody is humanized antibody, people's antibody or chimeric antibody.

46. the method for claim 45, wherein antibody is humanization 3D6 or humanization 12A11.

47. the method for claim 46, wherein humanized antibody is a Ba Biniu pearl monoclonal antibody (bapineuzumab).

48. the method for treatment Alzheimer's disease comprises

With reduce or suppress the blood vessel amyloidogenic become the pathology development, make little hemorrhage minimized and/or reduce or suppress the N-terminal that the dosage of A beta plaque development uses A β and have specific antibody.

49. the method for claim 48, wherein antibodies is in the residue of the 1-5 position of A β.

50. the method for claim 48, wherein antibody is humanized antibody, people's antibody or chimeric antibody.

51. the method for claim 50, wherein antibody is humanization 3D6 or humanization 12A11.

52. the method for claim 51, wherein humanized antibody is a Ba Biniu pearl monoclonal antibody (bapineuzumab).

53. the method for treatment Alzheimer's disease comprises

With reduce or suppress the blood vessel amyloidogenic become the pathology development, make little hemorrhage minimized and/or reduce or suppress the N-terminal that the dosage of neuritis's pathology development uses A β and have specific antibody.

54. the method for claim 53, wherein antibodies is in the residue of the 1-5 position of A β.

55. the method for claim 53, wherein antibody is humanized antibody, people's antibody or chimeric antibody.

56. the method for claim 55, wherein antibody is humanization 3D6 or humanization 12A11.

57. the method for claim 56, wherein humanized antibody is a Ba Biniu pearl monoclonal antibody (bapineuzumab).

58. the method for treatment Alzheimer's disease comprises

With reduce or suppress the blood vessel amyloidogenic become pathology, make little hemorrhage minimized and/or improve the N-terminal that the dosage of patient's cognitive function uses A β and have specific antibody.

59. the method for claim 58, wherein antibodies is in the residue of the 1-5 position of A β.

60. the method for claim 58, wherein antibody is humanized antibody, people's antibody or chimeric antibody.

61. the method for claim 60, wherein antibody is humanization 3D6 or humanization 12A11.

62. the method for claim 61, wherein humanized antibody is a Ba Biniu pearl monoclonal antibody (bapineuzumab).

63. each method of claim 48-62 wherein reduces or suppresses the blood vessel amyloidogenic to become pathology be to stop blood vessel A β accumulation or remove blood vessel A β.

64. be used for the treatment of the kit of CAA, comprise:

A. the vial that contains preparation; With

B. monitor the description of the patient's who uses described preparation CAA.

65. be used for the treatment of the kit of CAA, comprise:

A. the vial that contains preparation, described preparation comprise about 0.5 to 3mg/kg humanization anti-amyloid beta antibodies; With

B. monitor the description of the patient's who uses described preparation CAA, comprising:

I. the brain by MRI monitoring patient is little hemorrhage, or

Ii. monitor the removal of patient's blood vessel amyloid by PET scanning.

66. be used for the treatment of the kit of CAA, comprise:

A. the vial that contains preparation, described preparation comprises:

I. about 10mg is the humanization anti-amyloid beta antibodies of about 250mg extremely,

Ii. about 4% mannitol or about 150mM NaCl,

Iii. about 5mM to about 10mM histidine and

Iv. about 10mM methionine; And

B. monitor the description of the patient's who uses described preparation CAA, comprising:

I. the brain by MRI monitoring patient is little hemorrhage, or

Ii. monitor the removal of patient's blood vessel amyloid by PET scanning.

67. be used for the treatment of the kit of Alzheimer's disease, comprise:

A. the vial that contains preparation, described preparation comprises:

I. about 10mg is the humanization anti-amyloid beta antibodies of about 250mg extremely,

Ii. about 4% mannitol or about 150mM NaCl,

Iii. about 5mM to about 10mM histidine and

Iv. about 10mM methionine; And

B. monitor the description of the patient's who uses described preparation Alzheimer's disease, comprising:

I. the brain by MRI monitoring patient is little hemorrhage, or

Ii. monitor the removal of patient's blood vessel amyloid by PET scanning.

68. be used for the treatment of the kit of CAA and Alzheimer's disease, comprise:

A. the vial that contains preparation, described preparation comprises:

I. about 10mg is the humanization anti-amyloid beta antibodies of about 250mg extremely,

Ii. about 4% mannitol or about 150mM NaCl,

Iii. about 5mM to about 10mM histidine and

Iv. about 10mM methionine; And

B. monitor the patient's who uses described preparation the CAA and the description of Alzheimer's disease, comprising:

I. the brain by MRI monitoring patient is little hemorrhage, or

Ii. monitor the removal of patient's blood vessel amyloid by PET scanning.

69. the method for claim 2, thereby wherein antibody is according to being enough to make the average serum concentration of patient's antibody maintain also treatment patient's the scheme administration of 1-15 μ g antibody/ml serum.

70. the method for claim 69, wherein average serum concentration is in the scope of 1-10 μ g antibody/ml serum.

71. the method for claim 70, wherein average serum concentration is in the scope of 1-5 μ g antibody/ml serum.

72. the method for claim 71, wherein average serum concentration is in the scope of 2-4 μ g antibody/ml serum.

73. the method for claim 69, wherein antibody is through intravenous administration.

74. the method for claim 73, the wherein dosage of every month applied once 0.5-1.0mg/kg.

75. the method for claim 74, the wherein dosage of every month applied once 0.1-1.0mg/kg.

76. the method for claim 2, wherein antibody is through subcutaneous administration.

77. the method for claim 76, wherein antibody is with weekly extremely mensal frequency administration.

78. the method for claim 76, wherein antibody once in a week or administration biweekly whenever.

79. the method for claim 76, wherein antibody is with about dosed administration of 0.01 to about 0.35mg/kg.

80. the method for claim 76, wherein antibody is with about dosed administration of 0.05 to about 0.25mg/kg.

81. the method for claim 76, wherein antibody is weekly to administration biweekly whenever with about dosage of 0.015 to about 0.2mg/kg.

82. the method for claim 76, wherein antibody is weekly to administration biweekly whenever with about dosage of 0.05 to about 0.15mg/kg.

83. the method for claim 76, wherein antibody is with the weekly administration of about dosage of 0.05 to about 0.07mg/kg.

84. the method for claim 76, wherein antibody is with the weekly administration of the dosage of 0.06mg/kg.

85. the method for claim 76, wherein antibody is with the administration biweekly whenever of about dosage of 0.1 to about 0.15mg/kg.

86. the method for claim 2, wherein the average serum concentration of antibody kept 6 months at least.

87. the method for claim 2, wherein the average serum concentration of antibody kept at least one year.

88. the method for claim 2 also comprises the antibody concentration of measuring in the serum, and if the concentration that records be in outside the scope, then adjust scheme.

89. the method for claim 2, wherein antibody with about dosage of 0.01 to about 0.6mg/kg and weekly to mensal frequency through subcutaneous administration.

90. the method for claim 2, wherein antibody is with about dosed administration of 0.05 to about 0.5mg/kg.

91. the method for claim 89, wherein antibody is with about dosed administration of 0.05 to about 0.25mg/kg.

92. the method for claim 89, wherein antibody is weekly to administration biweekly whenever with about dosage of 0.015 to about 0.2mg/kg.

93. the method for claim 89, wherein antibody is weekly to administration biweekly whenever with about dosage of 0.05 to about 0.15mg/kg.

94. the method for claim 89, wherein antibody is with the weekly administration of about dosage of 0.05 to about 0.07mg/kg.

95. the method for claim 89, wherein antibody is with the weekly administration of the dosage of 0.06mg/kg.

96. the method for claim 89, wherein antibody is with the administration biweekly whenever of about dosage of 0.1 to about 0.15mg/kg.

97. the method for claim 89, wherein antibody is with every month single administration of about dosage of 0.1 to about 0.3mg/kg.

98. the method for claim 89, wherein antibody is with every month single administration of dosage of 0.2mg/kg.

99. the method for claim 2, wherein antibody is with about dosage of 1 to about 40mg and weekly to mensal frequency administration.

100. the method for claim 99, wherein antibody is with about dosed administration of 5 to about 25mg.

101. the method for claim 99, wherein antibody is with about dosed administration of 2.5 to about 15mg.

102. the method for claim 99, wherein antibody is weekly to administration biweekly whenever with about dosage of 1 to about 12mg.

103. the method for claim 99, wherein antibody is weekly to administration biweekly whenever with about dosage of 2.5 to about 10mg.

104. the method for claim 99, wherein antibody is with the weekly administration of about dosage of 2.5 to about 5mg.

105. the method for claim 99, wherein antibody is with the weekly administration of about dosage of 4 to about 5mg.

106. the method for claim 98, wherein antibody is with the administration biweekly whenever of about dosage of 7 to about 10mg.

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